Small Molecule Activators of Parkin Enzyme Function

ABSTRACT

The present disclosure relates to compounds for activating the enzymatic activity of an E3 ubiquitin ligase and methods for treating a disease or disorder in a subject with diminished E3 ubiquitin ligase enzymatic activity. In some embodiments, the present disclosure provides a compound of Formula I 
     
       
         
         
             
             
         
       
     
      or a compound of Formula II. 
     
       
         
         
             
             
         
       
     
      or pharmaceutically acceptable salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application Serial No.16/321,208, filed Jan. 28, 2019, which is a National Stage applicationunder U.S.C. § 371 of International Application No. PCT/US2017/044432,filed Jul. 28, 2017, which claims the benefit of U.S. ProvisionalApplication Serial No. 62/367,870, filed Jul. 28, 2016. The disclosuresof the prior applications are considered part of (and are incorporatedby reference in) the disclosure of this application.

TECHNICAL FIELD

The present disclosure relates to compounds for activating the enzymaticactivity of an E3 ubiquitin ligase and methods for treating a disease ordisorder in a subject with diminished E3 ubiquitin ligase enzymaticactivity.

BACKGROUND

The enzymatic activity of the broadly protective E3 ubiquitin ligasesand mitochondrial quality control is often lost or decreased in patientssuffering from certain diseases or disorders, such as Parkinson’sdisease, and is diminished during aging and in many other age-relatedhuman disorders. As a result, dysfunctional mitochondria accumulate andeventually result in cell death.

The mitochondrial kinase PINK1 and the cytosolic E3 ubiquitin ligaseParkin together mediate the selective degradation of damagedmitochondria by autophagy (mitophagy) (Narendra et al. (2008) J. CellBiol. 183:795-803; Geisler et al. (2010) Nat. Cell Biol. 12: 119-131).This crucial mitochondrial quality control pathway protects cells fromthe accumulation of harmful damaged mitochondria. While all cells areaffected by mitochondrial damage, energy-demanding cells like neuronsand muscle cells are especially vulnerable to failure of mitochondrialquality control. Loss-of function mutations of PINK1 and Parkin abrogatemitochondrial quality control and are associated with early-onsetrecessive Parkinson’s disease (PD) (Kitada et al. (1998) Nature392:605-608; Valente et al. (2004) Science 304:1158-1160). In addition,inactivation of Parkin has also been reported in sporadic, late-onset PD(Dawson et al. (2014) Neurodegener. Dis. 13:69-71; LaVoie et al. (2005)Nat. Med. 11:1214-1221; LaVoie et al. (2007) J. Neurochem.103:2354-2368; Wong et al. (2007) J. Biol. Chem. 282:12310-12318).However, PINK1 and Parkin are conserved amongst all multicellulareukaryotes and are widely expressed across all tissues/cells. Given thepresence of mitochondria in all cells and the need for such astress-induced quality control pathway, selective clearance throughmitophagy appears to be a fundamental, cytoprotective mechanism withfar-reaching implications beyond PD. Thus activation of Parkin has beenrecognized as a potentially beneficial and broadly applicable newtherapy for a wide-range of human diseases and aging.

SUMMARY

The foregoing and other aspects and embodiments of the disclosure can bemore fully understood by reference to the following detailed descriptionand claims.

The present application provides compounds of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   A is CH or O;-   B is CH or N;-   D is C or N;-   E is CH or N;-   W is C or N;-   X is C or N;-   Y is C or N;-   Z is C or N;-   R¹ is H, C₁₋₄ alkyl, phenyl, or hetAr¹;-   R² is H;-   R³ is H;-   or R² and R³, together with Y and X, form a 6-membered cycloalkyl    ring;-   R⁷ is H;-   or, optionally, when n is 0, R¹ and R⁷, together with the atoms to    which they are attached form a 6-membered cycloalkyl ring;-   R⁴ is H, C₁₋₄ alkyl, halogen, CF₃, or phenyl;-   R⁵ is H, C₁₋₄ alkyl, C₄₋₁₀ cycloalkyl, phenyl optionally substituted    with halogen, (C₁₋₃ alkyl)O(C₄₋₆ cycloalkyl), O(C₁₋₄ alkyl)(C₄₋₆    cycloalkyl), S(C₁₋₄ alkyl), S(C₄₋₆ cycloalkyl), (C₁₋₃ alkyl)(C₄-₉    hetCyc¹), hetAr¹, or O(phenyl) optionally substituted with CN;-   R⁶ is H or C₁₋₄ alkyl;-   hetAr¹ is a 6-membered heteroaryl ring having 1-3 ring nitrogen    atoms optionally substituted with C₁₋₄ alkyl;-   hetCyc¹ is a 6-10-membered bicyclic ring having at least one ring    heteroatom which is nitrogen and at least one of the rings is    aromatic;-   m is 0 or 1;-   n is 0 or 1;-   p is 0 or 1; and-   the dashed lines can be single or double bonds.

In some embodiments, A is CH.

In some embodiments, B is CH. In some embodiments, B is N.

In some embodiments, A is O and B is N.

In some embodiments, D is C. In some embodiments, D is N.

In some embodiments, E is CH. In some embodiments, E is N.

In some embodiments, W is N. In some embodiments, W is C.

In some embodiments, X is C.

In some embodiments, Y is C. In some embodiments, Y is N.

In some embodiments, Z is N. In some embodiments, Z is C.

In some embodiments, R¹ is H. In some embodiments, R¹ is C₁₋₄ alkyl orhetAr¹. In some embodiments, R¹ is methyl, isopropyl, or pyridine.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is Cl, CF₃, methylor phenyl.

In some embodiments, R⁵ is H or phenyl optionally substituted withhalogen. In some embodiments, R⁵ is (C₁₋₃ alkyl)O(C₄₋₆ cycloalkyl),O(C₁₋₄ alkyl)(C₄₋₆ cycloalkyl), or O(phenyl) which is optionallysubstituted with CN. In some embodiments, C₄₋₆ cycloalkyl is cyclopentylor cyclohexyl. In some embodiments, R⁵ is (C₁₋₃ alkyl)O(cyclopentyl) orO(C₁₋₄ alkyl)(C₄₋₆ cyclohexyl). In some embodiments, R⁵ is C₁₋₄ alkyl,C₄₋₁₀ cycloalkyl, or (C₁₋₃ alkyl)(C₄₋₉ hetCyc¹). In some embodiments,hetCyc¹ is a 9-membered bicyclic ring having one or more nitrogen atoms.In some embodiments, hetCyc¹ is isoindoline. In some embodiments, R⁵ isS(C₁₋₄ alkyl) or S(C₄₋₆ cycloalkyl). In some embodiments, C₄₋₆cycloalkyl cyclopentyl or cyclohexyl. In some embodiments, R⁵ is phenylsubstituted with F. In some embodiments, R⁵ is hetAr¹. In someembodiments, hetAr¹ is pyridine or pyrimidine optionally substitutedwith C₁₋₄ alkyl.

In some embodiments, R⁶ is H.

In some embodiments, m is 1.

In some embodiments, n is 0.

In some embodiment, p is 0. In some embodiments, p is 1.

In some embodiments, the compound of Formula I is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

The present application also provides compounds of Formula II:

or a pharmaceutically acceptable salt thereof, wherein:

-   A is CH, N, or S;-   B is CH, N, O, or S;-   D is C or N;-   E is CH or N;-   L is C₁₋₃ alkylene or C(═O);-   W is CH, CH₂, N, orNR^(a);-   Xis CH, CH₂, N, or NR^(b);-   Y is CH, CH₂ or O;-   Z is Nor CR^(2');-   R¹ is H or C₁₋₃ alkyl;-   R² is absent or C₁₋₆ alkylene;-   R^(2') is H or C₁₋₆ alkyl;-   or, when Z is CR^(2'), R² and R^(2'), together with C, can be taken    together to form a C₁₋₆ heterocyclic ring;-   R³ is H, halogen, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, or C₁₋₃ alkoxy;-   R⁴ is H or C₁₋₃ alkyl;-   R^(4') is H or C₁₋₃ alkyl;-   R^(a) is H or C₁₋₃ alkyl;-   R^(b) is C₁₋₃ alkyl;-   n is 1 or 2;-   m is 0, 1 or 2;-   p is 0 or 1; and-   the dashed lines can be single or double bonds;-   with the proviso that when n is 2, R¹ is methyl, D is C, R³ is H,    and A, B, and E are all CH, then at least one of W, X, and Y is not    CH₂.

In some embodiments, A is CH. In some embodiments, A is N. In someembodiments, A is S.

In some embodiments, B is CH. In some embodiments, B is N, O, or S.

In some embodiments, D is C.

In some embodiments, E is CH. In some embodiments, E is N.

In some embodiments, L is C₁₋₃ alkylene. In some embodiments, L ismethylene.

In some embodiments, W is CH₂. In some embodiments, W is N.

In some embodiments, X is CH₂. In some embodiments, X is CH. In someembodiments, X is NR^(b). In some embodiments, R^(b) is methyl.

In some embodiments, Y is CH₂. In some embodiments, Y is CH.

In some embodiments, Z is N.

In some embodiments, R² is absent.

In some embodiments, Z is CR²'. In some embodiments, R² and R^(Z'),together with C, form a 5-membered heterocyclic ring having 1 nitrogenatom.

In some embodiments, R¹ is H. In some embodiments, R¹ is methyl.

In some embodiments, R^(a) is H or methyl.

In some embodiments, R^(b) is methyl.

In some embodiments, R³ is H, halogen, or C₃₋₆ cycloalkyl. In someembodiments, R³ is chlorine. In some embodiments, R³ is cyclopropyl.

In some embodiments, R⁴ and R^(4') are each H. In some embodiments, R⁴and R^(4') are each methyl.

In some embodiments, n is 2. In some embodiments, n is 1.

In some embodiments, m is 1 or 2.

In some embodiments, p is 0. In some embodiments, p is 1.

In some embodiments, the compound of Formula II is selected from thegroup consisting of:

and

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula II is a compound of FormulaIIa:

or a pharmaceutically acceptable salt thereof, wherein:

-   A is CH or N;-   B is N, O, or S;-   D is C or N;-   E is CH or N;-   W is CH2, NR^(a);-   X is CH, CH₂, N, or NR^(b);-   Y is CH, CH₂ or O;-   L is C₁₋₃ alkylene or C(═O);-   R¹ is H or C₁₋₃ alkyl;-   R³ is C₁₋₃ alkyl or C₁₋₃ alkoxy;-   R⁴ is H or C₁₋₃ alkyl;-   R^(4') is H or C₁₋₃ alkyl;-   R^(a) is H or C₁₋₃ alkyl;-   R^(b) is C₁₋₃ alkyl;-   n is 1 or 2;-   m is 0, 1 or 2;-   p is 0 or 1; and-   the dashed lines can be single or double bonds.

In some embodiments, the compound of Formula II is a compound of FormulaIIb:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is C₁₋₃ alkyl; and-   R⁵ is (C₁₋₃ alkyl)phenyl.

The present application further provides pharmaceutical compositionscomprising a compound provided herein, or a pharmaceutically acceptablesalt thereof, and at least one pharmaceutically acceptable carrier.

The present application further provides methods of activating theenzymatic activity of an E3 ubiquitin ligase in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound provided herein, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the E3 ubiquitin ligase is selected from the groupconsisting of Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31 (HOIP), RBCK1(HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2.

In some embodiments, the enzymatic activity is activated or enhancedduring mitochondrial stress.

In some embodiments, the compound stimulates mitochondrial qualitycontrol.

In some embodiments, the compound interferes with the auto-inhibition ofthe ligase.

In some embodiments, the subject has diminished E3 ubiquitin ligaseenzymatic activity. In some embodiments, the enzymatic activity isdiminished due to a disease, aging, or an age-related disorder.

In some embodiments, the disease or disorder is selected from the groupconsisting of Parkinson’s disease, parkinsonism, Alzheimer’s disease,dementia, Amyotrophic lateral sclerosis, Frontotemporal dementia,autism, depression, progeroid disorder, leprosy, an inclusion bodymyositis, diabetes mellitus, diabetic kidney disease, a liver disease, alysosomal storage disorder, a neurological disease, a muscular disease,a mitochondrial disease, and cancer.

In some embodiments, the disease or disorder is Parkinson’s disease.

In some embodiments, the disease or disorder is cancer. In someembodiments, the cancer is selected from the group consisting of livercancer, brain cancer, skin cancer, kidney cancer, lung cancer, coloncancer, pancreatic cancer, hepatocellular carcinoma, glioma, skincutaneous melanoma, clear cell renal cell carcinoma, non-small cell lungcancer, lung adenocarcinoma, lung squamous cell cancer, colorectalcancer, pancreatic adenocarcinoma, adenoid cystic carcinoma, acutelymphoblastic leukemia, chronic myeloid leukemia, bladder urothelialcancer, head and neck squamous cell carcinoma, esophagealadenocarcinoma, gastric cancer, cervical cancer, thyroid cancer, andendometrioid cancer.

The present application further provides a method of treating a diseaseor disorder associated with diminished E3 ubiquitin ligase enzymaticactivity in a subject, the method comprising administering to thesubject a therapeutically effective amount of a compound providedherein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the E3 ubiquitin ligase is selected from the groupconsisting of Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31 (HOIP), RBCK1(HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2.

In some embodiments, the disease or disorder is selected from the groupconsisting of Parkinson’s disease, parkinsonism, Alzheimer’s disease,dementia, Amyotrophic lateral sclerosis, Frontotemporal dementia,autism, depression, progeroid disorder, leprosy, an inclusion bodymyositis, diabetes mellitus, diabetic kidney disease, a liver disease, alysosomal storage disorder, a neurological disease, a muscular disease,a mitochondrial disease, and cancer.

In some embodiments, the disease or disorder is Parkinson’s disease.

In some embodiments, the disease or disorder is cancer. In someembodiments, the cancer is selected from the group consisting of livercancer, brain cancer, skin cancer, kidney cancer, lung cancer, coloncancer, pancreatic cancer, hepatocellular carcinoma, glioma, skincutaneous melanoma, clear cell renal cell carcinoma, non-small cell lungcancer, lung adenocarcinoma, lung squamous cell cancer, colorectalcancer, pancreatic adenocarcinoma, adenoid cystic carcinoma, acutelymphoblastic leukemia, chronic myeloid leukemia, bladder urothelialcancer, head and neck squamous cell carcinoma, esophagealadenocarcinoma, gastric cancer, cervical cancer, thyroid cancer, andendometrioid cancer.

The present application further provides a method of treating a diseaseor disorder in a subject, the method comprising:

-   (a) detecting a disease or disorder associated with diminished E3    ubiquitin ligase enzymatic activity; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, or a pharmaceutically acceptable salt    thereof.

In some embodiments, the E3 ubiquitin ligase is selected from the groupconsisting of Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31 (HOIP), RBCK1(HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2.

In some embodiments, the disease or disorder is selected from the groupconsisting of Parkinson’s disease, parkinsonism, Alzheimer’s disease,dementia, Amyotrophic lateral sclerosis, Frontotemporal dementia,autism, depression, leprosy, an inclusion body myositis, diabetesmellitus, diabetic kidney disease, a liver disease, a lysosomal storagedisorder, a neurological disease, a muscular disease, a mitochondrialdisease, and cancer.

In some embodiments, the disease or disorder is Parkinson’s disease.

In some embodiments, the disease or disorder is cancer. In someembodiments, the cancer is selected from the group consisting of livercancer, brain cancer, skin cancer, kidney cancer, lung cancer, coloncancer, pancreatic cancer, hepatocellular carcinoma, glioma, skincutaneous melanoma, clear cell renal cell carcinoma, non-small cell lungcancer, lung adenocarcinoma, lung squamous cell cancer, colorectalcancer, pancreatic adenocarcinoma, adenoid cystic carcinoma, acutelymphoblastic leukemia, chronic myeloid leukemia, bladder urothelialcancer, head and neck squamous cell carcinoma, esophagealadenocarcinoma, gastric cancer, cervical cancer, thyroid cancer, andendometrioid cancer.

The present application further provides a method of treatingParkinson’s disease in a subject, the method comprising:

-   (a) detecting Parkinson’s disease in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, or a pharmaceutically acceptable salt    thereof.

The present application further provides a method of treating anage-related disorder in a subject, the method comprising:

-   (a) detecting an age-related disorder in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, or a pharmaceutically acceptable salt    thereof.

The present application further provides a method of treating cancer ina subject, the method comprising:

-   (a) detecting cancer in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, or a pharmaceutically acceptable salt    thereof.

In some embodiments, the cancer is selected from the group consisting ofliver cancer, brain cancer, skin cancer, kidney cancer, lung cancer,colon cancer, pancreatic cancer, hepatocellular carcinoma, glioma, skincutaneous melanoma, clear cell renal cell carcinoma, non-small cell lungcancer, lung adenocarcinoma, lung squamous cell cancer, colorectalcancer, pancreatic adenocarcinoma, adenoid cystic carcinoma, acutelymphoblastic leukemia, chronic myeloid leukemia, bladder urothelialcancer, head and neck squamous cell carcinoma, esophagealadenocarcinoma, gastric cancer, cervical cancer, thyroid cancer, andendometrioid cancer.

The present application further provides a method of activating theenzymatic activity of an E3 ubiquitin ligase in a cell, the methodcomprising contacting the cell with a compound provided herein, or apharmaceutically acceptable salt thereof.

In some embodiments, the E3 ubiquitin ligase is selected from the groupconsisting of Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31 (HOIP), RBCK1(HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2.

In some embodiments, the enzymatic activity is activated duringmitochondrial stress.

In some embodiments, the compound stimulates mitochondrial qualitycontrol.

In some embodiments, the compound interferes with the auto-inhibition ofthe ligase.

In some embodiments, the cell has diminished E3 ubiquitin ligaseenzymatic activity.

In some embodiments, the contacting is in vitro.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the results of a primary screening assay with highcontent imaging (HCI) of enhanced green fluorescent protein(EGFP)-Parkin translocation. FIG. 1A shows the results of the HCI assayusing HeLa cells stably expressing GFP-Parkin. GFP-Parkin is localizedthroughout the cell in unstressed cells but translocates to mitochondriawhen the mitochondrial membrane potential is compromised and Parkin isactivated. After automated image acquisition of fixed cells, regions ofinterest of each cell were defined with the analysis software. The assayused a “RING — two output” algorithm that resulted in a nuclear mask anda cytoplasmic mask that were being created with help of the nuclear dyeHoechst. To quantify the relocalization of GFP-Parkin to mitochondria,the ratio of the GFP intensity in the cytoplasm and the nucleus wascalculated. Under non-stressed condition, this ratio is about 1. FIG. 1Bis a bar graph showing the results of the assay with a Z factor (Z') of0.5-0.8, which indicates its robustness and usefulness as a screeningassay. Validation of the assay has been performed by using PINK1 siRNA.

FIGS. 2A-2C show a primary HCI assay with CCCP titration. FIG. 2A is agraphic description of the primary HCI assay of EGFP-Parkintranslocation. HeLa cells stably expressing GFP-Parkin were seeded with1350 cells per well in 384-well plates (25 µL per well). Cells wereallowed to attach overnight before drug was added 2x concentrated in avolume of 25 µL in triplicates. After 2 h, 50 µL of a 2x low-dose CCCP(yielding a final concentration of 3.5 µM CCCP) was added to all wellsthat contained test compounds and for the negative control. For positivecontrol wells, CCCP was added in a final concentration of 10 µM. After 2h incubation with CCCP, cells were fixed and labeled with Hoechst for atleast 1 h before cells were imaged. FIG. 2B shows an example of a CCCPtitration. FIG. 2C shows the result of a typical drug screen plate.Values were being normalized to negative and positive control values andplotted as % activity. Z' was calculated for quality control. Positivecontrols were indicated in green, negative control wells in red. Drugsthat lead to more than 50% activity are considered positive (blue).

FIGS. 3A-3E show the dose-response curves (DRCs) of Compounds 1 (FIG.3B), 2 (FIG. 3C), 3 (FIG. 3D), 4 (FIG. 3E), and 31 (FIG. 3A), whichshowed activity at 1 µM. The graphs show DRCs of EGFP-Parkintranslocation to damaged mitochondria. Cells were treated in 384-wellplates in triplicates with 12 different concentrations of the drugs. %activity is plotted versus the concentration [M] in log scale. Curve fitand EC₅₀ value calculations were done with Graph Pad Prism software. AllEC_(50S) are in the upper nanomolar range.

FIGS. 4A-4E show normalized DRCs of Compounds 1 (FIG. 4B), 2 (FIG. 4C),3 (FIG. 4D), 4 (FIG. 4E), and 31 (FIG. 4A) with pS65-Ub antibodies(EGFP-Parkin translocation vs. pS65-UB levels vs. cell death). Thenumber of cells are indicated by squares. The curve fit of pS65-Ub isindicated by circles. The curve fit of translocation is indicated bytriangles. Cell death was controlled for by calculating the number ofcells per well. In addition, the same plates were stained with pS65-Ubantibodies. Overlay of normalized DRCs of Parkin translocation andpS65-Ub signal gave similar values for all tested compounds.

FIG. 5 shows enhanced Ub-charging of FLAG-Parkin C431S (WB) bypre-treatment with Compounds 1, 2, 3, 4, and 31. Another measure ofParkin activation is Ub charging of Parkin. Parkin receives Ub from anE2 enzyme to transfer it to a substrate protein. The Ub is bound byParkin C431 in an unstable thioester bond. A serine substitution of thisamino acid (C431S) leads to a stable oxyester bond. The bound Ub canthen be visualized as a band shift in Western blot experiments. HeLa3xFLAG-Parkin C431S cells were seeded in 12-well plates and allowed toattach overnight. Cells were treated with 5 µM (top panels) or 1 µM(bottom panels) of Compounds 1, 2, 3, 4, and 31 2 h before adding CCCPfor another 2 h. 10µM CCCP was added to positive control (PC) wellswhereas all other samples received a low dose concentration of 3.5 µM.Cells were harvested in boiling hot SDS lysis buffer and proteinconcentration was determined by BCA. Samples were split and left eitheruntreated or were treated with NaOH as indicated. Samples were run on an8-16% Tris-Glycine gel, blotted onto membranes and probed withantibodies against Flag and pS65-Ub. GAPDH served as a loading control.Band shift indicates Parkin binding to Ubiquitin, which is cleavablewith NaOH.

FIG. 6 shows enhanced Ub-charging of FLAG-Parkin C431S (MSD assay) bypre-treatment with Compounds 1, 2, 3, 4, and 31. Ubiquitin charging ofParkin was monitored by ELISA-like MesoScale Discovery assay that useselectrochemiluminescence. Plates were coated with Flag antibody andincubated with lysates from 3xFLAG-Parkin C431S cells that had beenpretreated for 2 h with 5 µM of Compounds 1, 2, 3, 4, and 31 or DMSO(left, -) before incubating them for another 2 h with (+) or without (-)low-dose (3.5 µM) CCCP. Positive control (PC) cells were treated with 10µM CCCP. After washing, pS65-Ub antibody was added together with asulfo-tagged anti-rabbit antibody. Values were normalized to the PC(10µM CCCP) and negative control (-). Statistical analysis was performedby one-way ANOVA with Tukey’s post-hoc test. ****, p<0.0001.

FIG. 7 shows enhanced ubiquitination and degradation of Parkinsubstrates by pre-treatment with Compounds 1, 2, 3, 4, and 31. HeLacells stably expressing untagged Parkin were left untreated orpre-treated with 5 µM of Compounds 1, 2, 3, 4, and 31 for 2 h andtreated without (-) or with (+) low dose CCCP (3.5 µM CCCP). As apositive control (PC), some cells were treated with 10 µM CCCP. Lysateswere loaded onto 8-16% Tris-Glycine gels, blotted onto membranes andprobed with antibodies against Parkin substrates. While low-dose CCCPtreatment alone does not lead to substrate degradation, cells that werepre-treated with Compounds 1, 2, 3, 4, and 31 showed reduced substratelevels, similar to positive control cells.

FIG. 8 shows enhanced amplification of pS65-Ub signal by pre-treatmentwith Compounds 1, 2, 3, 4, and 31. HeLa cells stably expressing untaggedParkin were left untreated or pre-treated with 5 µM of Compounds 1, 2,3, 4, and 31 for 2 h and treated without (-) or with (+) low dose CCCP(3.5 µM CCCP). The positive control (PC) was treated with 10 µM CCCP for2 h. Lysates were loaded onto 8-16% Tris-Glycine gels, blotted ontomembranes and probed with antibodies against pS65-Ub. While low-doseCCCP treatment alone does not lead to pS65-Ub accumulation, cellstreated with Compounds 1, 2, 3, 4, and 31 showed robust induction ofpS65-Ub to the positive control.

FIG. 9 shows enhanced mitophagy flux upon pre-treatment with compound 4in the presence of mitochondrial damage. HCI in 384-well plates was usedto calculate the ratio of acidic to neutral mtKeima. Upon treatment with10 µM CCCP, there was significant increase of the mtKeima ratio.Pre-treatment with compound 4 (dashed line, here shown 5 µM of compound4) induced this also at low-dose CCCP concentrations while low dose CCCPalone (solid line) had only little effect on the ratio of acidic toneutral mtKeima.

FIG. 10 shows DRCs of Compounds 1, 2, 3, 4, and 31 in a mitophagy assay.HeLa mtKeima cells were pre-treated for 2 h with 12 different doses ofCompounds 1, 2, 3, 4, and 31 before low-dose CCCP was added (3 µM finalconcentration). 10 µM CCCP was added to positive control wells. Cellswere imaged live after 4 h and 8 h of CCCP. Values were normalized tothe positive and negative (3 µM CCCP) control values for each timepoint. Curve fitting was used to calculate EC₅₀ values.

FIG. 11 shows quality control of mitochondrial membrane potential (JC-10assay) upon treatment with Compounds 1, 2, 3, 4, and 31. In order toexclude compounds that diminish the mitochondrial membrane potential, aJC-10 assay was used. HeLa cells were plated in 384-well plates andtreated with different doses of CCCP or with 5 µM of Compounds 1, 2, 3,4, and 31 as indicated for 2 h. The JC-10 dye was added to the livecells and cells are stained for 45 minutes. The plates were thenmeasured with a fluorescent plate reader. JC-10 is a mitochondrial dyethat emits red fluorescence in the presence of mitochondrial membranepotential. The fluorescence will change to green in the absence ofmitochondrial membrane potential. While Compounds 1, 2, 3, and 4 did notshow an effect on JC-10, Compound 31 significantly increaseddepolarization of mitochondria compared to the DMSO control. Shown areaverage values of 3 independent experiments. Statistical analysis wasperformed by one-way ANOVA with Tukey’s post-hoc test. ***, p< 0.0005.

FIG. 12 is a summary of parameters of Compounds 1-6,8,20, and 31. Thistable summarizes chemoinformatic properties and in vitro toxicityparameters of 20 PACs together with their molecular weight and theirEC₅₀ values as assessed in the primary assay screen (Parkintranslocation to mitochondria). Compounds show zero violations ofLipinski’s Rule of Five, are neutral on CNS metric or even predicted forgood CNS penetrance with low in silico toxicity concerns, as well asexcellent MW, PSA, logP, and Caco-2 for improved CNS function.

FIG. 13 shows the normalized EGFP-Parkin translocation [%] in responseto different CCCP doses.

FIG. 14 shows HeLa EGFP-Parkin cells seeded in 384-well plates andtreated with 3.5 µM CCCP as the negative control and 10 µM CCCP for thepositive control. Cells were either pretreated for 2 h with compounds 1,2, 3, 4, or 31 before low dose CCCP was added or the compound and CCCPwere added at the same time. Cells were fixed and analyzed with HCI forParkin translocation. Both experimental regimens led to similar EC₅₀values for all five compounds.

FIGS. 15A-15H show Parkin activation in primary fibroblasts, neuronalcells and in vitro. Positive control (PC) cells were treated with DMSOand 10 µM of CCCP. Negative control (NC) cells were treated with 3.5 µMCCCP, some cells were left untreated (-). FIG. 15A: Western blot ofhuman fibroblasts treated with 5 µM of compound 4 for 2 h beforedifferent concentrations of CCCP were added shows increasedubiquitylation/degradation of mitofusins and amplification of thepSer65-Ub signal. Vinculin was used as loading control. FIG. 15B:Western blot of fibroblasts pre-treated with 5 µM of compounds 1, 2, 3,4, and 31 before low dose CCCP (3.5 µM) show enhanced degradation of thesubstrates MFN1/2 and increase of pSer65-Ub levels. FIG. 15C: Humanfibroblasts were directly converted to neurons and treated as in FIG.15B. Western blot shows the induction of modified MFN1 and of pSer65-Ubupon compound treatment, similar to PC, but not NC cells. Beta IIItubulin confirmed successful conversion of fibroblasts to iNeurons. FIG.15D: Rat PC12 cells were treated as in FIG. 15A. Western blots showenhanced ubiquitylation of MFN2 and TOM70, and robust pSer65-Ubinduction similar to PC, but not NC cells. FIG. 15E: In vitro E2discharge assay. Recombinant Parkin was pre-incubated with compound 4 orDMSO as control and was mixed to Ub-loaded UbcH7. FLAG-Ubiquitin andUbcH7 western blots both show slightly more E2 discharge in presence ofcompound 4. Control reactions were performed without PINK1 or Parkin.FIG. 15F: In vitro assay with isolated mitochondria from untreated orCCCP-treated HeLa cells lacking Parkin. Mitochondrial preparations weremixed with recombinant Parkin, E1, UbcH7, ATP and either compound 4 orDMSO as a control and incubated for the indicated times. MFN1 westernblot show increased ubiquitylation when compound 4 is present. Controlsamples were incubated in reaction mix without Parkin. FIG. 15G: Proteinmelting was performed with 50 ng purified Parkin mixed with SYPRO orangeand DMSO as control or 500 nM compound 4. Analysis of triplicatereactions revealed a 3° C. temperature shift. FIG. 15H: Statisticalanalysis of three independent experiments as performed in FIG. 15G.Shown in the average value +/- SD (unpaired, two-sided t-test; ***p<0.0005).

FIGS. 16A-16D shows effects in primary and neuronal cells. FIG. 16A:Quantification of pSer65-Ub signal using sandwich ELISA from sixindependent experiments performed in human fibroblast treated asdescribed in FIG. 15A. Shown in the average -/+ SD. One-way ANOVA withTukey’s posthoc test (*** p< 0.0005). FIG. 16B: Human primaryfibroblasts were treated with different concentrations of all fivecompounds (1, 2, 3, 4, and 31) for 2 h. Compound treatment alone (i.e.,in the absence of CCCP) did not induce MFN1 ubiquitylation or pSer65-Ubsignals. Different CCCP concentrations were used as positive control.FIG. 16C: Rat PC12 cells were pretreated with 5 µM of the compounds for2 h and then treated with low dose of CCCP (3.5 µM). Control cells weretreated with DMSO and either 10 µM CCCP (PC) or 3.5 µM (NC). Some cellsreceived no CCCP (-). Western blot shows ubiquitinylation (grayarrowheads) and or decrease of unmodified (black arrowhead) MFN1/2 andTOM70. The PINKl/Parkin product pSer65-Ub was induced by all fivecompounds upon low dose treatment and in positive controls. FIG. 16D:PC12 cells were differentiated by treatment with NGF (NGF+). Some cellswere left undifferentiated (NGF-). Cells were treated with 5 µM ofcompounds or DMSO as indicated. 2 h later, low dose CCCP (3.5 µM) wasadded to cells treated with test compounds. DMSO controls were eithertreated with 3.5 µM CCCP (NC) or 10 µM CCCP (PC). Some cells were nottreated with CCCP (-). Western blots probed with antibodies against MFN1show degradation in cells treated with compounds and in the positivecontrol. Some degradation can also be observed in the negative control.The PINKl/Parkin product pSer65 shows increased induction in cells thathave been treated with compound compared to the negative control. BetaIII tubulin was used to confirm successful differentiation. Vinculin wasused as loading control.

FIGS. 17A-17L are detailed chemoinformatic properties of the Parkinactivating compounds 1-6, 8-12, 14-20, and 29-31. The table lists thecompounds in columns with each column containing the docking score,experimental activity (nM) from dose response, chemoinformaticsproperties, and ligand efficiency. FIGS. 17A-17C show properties forcompounds 1-4 and 6. FIGS. 17D-17F show properties for compounds 8-12.FIGS. 17G-17I show properties for compounds 14-18. FIGS. 17J-17L showproperties for compounds 19, 20, and 29-31.

FIG. 18 is a bar graph showing tracer binding inhibition from in vitrohERG fluorescence polarization assay for compounds 1-4 and 31.

FIGS. 19A-19D show microsomal stability data for compounds 1-4 and 31and reference compounds (imipramine and propranolol) in mouse livermicrosomes.

FIGS. 20A-20E are the CYP inhibition profiles for compounds 1-4 and 31.FIG. 20A is the CYP inhibition profile for compound 31. FIG. 20B is theCYP inhibition profile for compound 1. FIG. 20C is the CYP inhibitionprofile for compound 2. FIG. 20D is the CYP inhibition profile forcompound 3. FIG. 20E is the CYP inhibition profile for compound 4.

FIG. 21 shows enhanced Ub-charging of FLAG-Parkin C431S (WB) bypre-treatment with compounds 5, 21-28, 32, 33, and 43-46. Band shiftindicates Parkin binding to Ubiquitin, which is cleavable with NaOH.

DETAILED DESCRIPTION

Parkin is a cytosolic E3 ubiquitin (Ub) ligase and acts downstream ofthe mitochondrial kinase PINK1. Upon mitochondrial stress, PINK1 isstabilized on the outer mitochondrial membrane and recruits Parkin byphosphorylation of Ub at the conserved residue Serine 65 (Kane et al.(2014) J. Cell Biol. 205:143-153; Kazlauskaite et al. (2014) Biochem. J.460:127-139; Koyano et al. (2014) Nature 510:162-166). Phosphorylated Ub(pS65-Ub) can activate Parkin and also acts as the receptor for Parkinon the mitochondrial surface (Okatsu et al. (2015) J. Cell Biol.109:111-128). Re-localization of Parkin is associated with its enzymaticactivation and the ligation of Ub molecules onto mitochondrial substrateproteins (Kazlauskaite et al. (2014) Open Biol. 4:130213) that in turnserve as additional substrates for PINK1 and Parkin (Fiesel et al.(2015) J. Cell Sci. 127:3488-3504). The formed pS65-Ub signal acts asthe mitophagy tag and is recognized by autophagy adapters for eventualdegradation of the whole organelle in the lysosome (Ordureau et al.(2015) Proc. Natl. Acad. Sci. U.S.A. 112:6637-6642; Richter et al.(2016) Proc. Natl. Acad. Sci. U.S.A. 113:4039-4044).

Upon stress, the PINKl/Parkin pathway promotes turnover of mitochondriaand prevents the accumulation of dysfunctional mitochondria that canlead to cellular degeneration. Under basal conditions, both PINK1 andParkin are repressed through different mechanisms. PINK1 isconstitutively cleaved by the mitochondrial protease PARL andsubsequently degraded by the proteasome (Yamano et al. (2013) Autophagy9:1758-1769). Parkin is present under basal conditions but isstructurally very compact with several self-interactions that prohibitactivity (Caulfield et al. (2015) Biochem. Soc. Trans. 43:269-274;Caulfield et al. (2014) PLoS Comput. Biol. 10:e1003935). Theseself-interactions have to be released and Parkin needs to ‘open up’ inorder to become active. Parkin is a RING-in-between-RING (RBR) ligase, arecently described new family of E3 ubiquitin ligases (Wenzel et al.(2011) Nature 474:105-108). Like members of classical RING-type ligases,it contains several RING domains that bind the E2 ubiquitin-conjugatingco-factors. Mechanistically however, Parkin acts like a HECT E3 ligaseas it physically receives the Ub moiety with its active cysteine (C431)from the E2 before it is transferred onto a substrate. Ub charging ofParkin (i.e., activation) is intimately linked to its mitochondrialrecruitment (Iguchi et al. (2013) J. Biol. Chem. 288:22019-22032; Zhenget al. (2013) Cell Res. 23:886-897). Parkin contains an N-terminalubiquitin-like (UBL) domain with a conserved Ser65 residue. Togetherwith phosphorylation of the modifier protein Ub at S65, PINK1-dependentphosphorylation of Parkin S65 within the UBL (Kazlauskaite et al. (2014)Biochem. J. 460:127-139; Iguchi et al. (2013) J. Biol. Chem.288:22019-22032; Shiba-Fukushima et al. (2014) PLoS Genet. 10:e1004391)is a key event leading to Parkin activation (Kazlauskaite et al. (2014)Open Biol. 4:130213; Caulfield et al. (2014) PLoS Comput. Biol.10:e1003935).

Accordingly, the present application provides compounds useful foractivating Parkin. These Parkin activating compounds (PACs) are activein the low micromolar / high nanomolar range and have been extensivelytested in human cell culture. The purpose of these compounds is toimprove PINK1/Parkin mitochondrial quality control. This pathway isimpaired in different forms of familial and sporadic PD. In addition,activation of mitochondrial quality control may prove beneficial for avariety of neurological, muscular and other age-related diseases.

In contrast to inhibitors of an enzyme/pathway that often require morethan 90% activity (i.e., inhibition), activators/enhancers of a givenenzyme/pathway may only require 10% activity (i.e., activation), sinceeven small amounts of active target can be further amplified along thepathway. This decreases side effects due to lower drug concentrationsneeded for effectiveness. In some embodiments, the herein described PACscan enhance Parkin activation upon an initial mitochondrialstress-induced phosphorylation mediated by PINK1 that results inconformational changes of Parkin and allows access to the drug-bindingsite. Without being bound by any theory, it is believed that this canlimit activation of Parkin’s enzymatic functions only when and whereneeded.

1. Definitions

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom, usually a carbon, oxygen, or nitrogenatom, is replaced with a selection from the indicated group, providedthat the designated atom’s normal valency is not exceeded, and that thesubstitution results in a stable compound. When a substituent is keto oroxo (i.e., ═O), then 2 hydrogens on the atom are replaced. Ring doublebonds, as used herein, are double bonds that are formed between twoadjacent ring atoms (e.g., C═C, C═N, N═N, etc.).

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁₋₄ alkyl is intended toinclude Ci, C₂, C₃, and C₄. C₁₋₆ alkyl is intended to include C₁, C₂,C₃, C₄, C₅, and C₆ alkyl groups and C₁₋₈ alkyl is intended to includeCi, C₂, C₃, C₄, C₅, C₆, C₇, and C₈. Some examples of alkyl include, butare not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl, n-hexyl, n-heptyl, and n-octyl.

As used herein, “alkenyl” is intended to include hydrocarbon chains ofeither straight or branched configuration and one or more unsaturatedcarbon-carbon bond that can occur in any stable point along the chain,such as ethenyl and propenyl. For example, C₂₋₆ alkenyl is intended toinclude C₂, C₃, C₄, C₅, and C₆ alkenyl groups and C₂-₈ alkenyl isintended to include C₂, C₃, C₄, C₅, C₆, C₇, and C₈ alkenyl groups.

As used herein, “alkylene” is intended to include moieties which arediradicals, i.e., having two points of attachment. A non-limitingexample of such an alkylene moiety that is a diradical is —CH₂CH₂—,i.e., a C₂ alkyl group that is covalently bonded via each terminalcarbon atom to the remainder of the molecule. The alkylene diradicalsare also known as “alkylenyl” radicals. Alkylene groups can be saturatedor unsaturated (e.g., containing —CH═CH— or —C═C— subunits), at one orseveral positions. In some embodiments, alkylene groups include 1 to 9carbon atoms (for example, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or1 to 2 carbon atoms). Some examples of alkylene groups include, but arenot limited to, methylene, ethylene, n-propylene, iso-propylene,n-butylene, iso-butylene, sec-butylene, tert-butylene, n-pentylene,iso-pentylene, sec-pentylene and neo-pentylene.

As used herein, “cycloalkyl” is intended to include saturated orunsaturated nonaromatic ring groups, such as cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. For example, the term “C₃₋₈ cycloalkyl” isintended to include C₃, C₄, C₅, C₆, C₇, and C₈ cycloalkyl groups.Cycloalkyls may include multiple spiro- or fused or bridged rings. Forexample, cycloalkyl can include, but is not limited to, spiro butyl,pentyl, hexyl, heptyl, octyl, nonyl, or decyl groups, bicyclo butyl,pentyl, hexyl, heptyl, octyl, nonyl, or decyl groups, adamantyl groups,and norbomyl groups.

As used herein, the term “heterocycloalkyl” refers to a saturated orunsaturated nonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic(fused, bridged, or spiro rings), or 11-14 membered tricyclic ringsystem (fused, bridged, or spiro rings) having one or more heteroatoms(such as O, N, S, or Se), unless specified otherwise. A heterocycloalkylgroup containing a fused aromatic ring can be attached through anyring-forming atom including a ring-forming atom of the fused aromaticring. In some embodiments, the heterocycloalkyl is a monocyclic 4-6membered heterocycloalkyl having 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur and having one or moreoxidized ring members. In some embodiments, the heterocycloalkyl is amonocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or4 heteroatoms independently selected from nitrogen, oxygen, or sulfurand having one or more oxidized ring members. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl,indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl,thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,1,4-dioxa-8-azaspiro[4.5]decanyl and the like.

As used herein, “amine” or “amino” refers to unsubstituted —NH₂ unlessotherwise specified.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, andiodo substituents.

As used herein, “haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with one or more halogen(for example -C_(v)F_(w)H_(2v-) _(w) ₊₁ wherein v = 1 to 3 and w = 1 to(2v+1)). Examples of haloalkyl include, but are not limited to,trifluoromethyl, trichloromethyl, pentafluoroethyl, andpentachloroethyl.

The term “haloalkoxy” as used herein refers to an alkoxy group, asdefined herein, which is substituted one or more halogen. Examples ofhaloalkoxy groups include, but are not limited to, trifluoromethoxy,difluoromethoxy, pentafluoroethoxy, trichloromethoxy, etc.

As used herein, “alkoxyl” or “alkoxy” refers to an alkyl group asdefined above with the indicated number of carbon atoms attached throughan oxygen bridge. C₁₋₆ alkoxy, is intended to include C₁, C₂, C₃, C₄,C₅, and C₆ alkoxy groups. C₁₋₈ alkoxy, is intended to include C₁, C₂,C₃, C₄, C₅, C₆, C₇, and C₈ alkoxy groups. Examples of alkoxy include,but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

As used herein, “aryl” includes groups with aromaticity, including“conjugated,” or multicyclic systems with at least one aromatic ring anddo not contain any heteroatom in the ring structure. Aryl may bemonocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term“C_(n)-_(m) aryl” refers to an aryl group having from n to m ring carbonatoms. In some embodiments, aryl groups have from 6 to 10 carbon atoms.In some embodiments, the aryl group is phenyl or naphthyl.

As used herein, the terms “aromatic heterocycle,” “aromaticheterocyclic” or “heteroaryl” ring are intended to mean a stable 5, 6,7, 8, 9, 10, 11, or 12-membered monocyclic or bicyclic aromatic ringwhich consists of carbon atoms and one or more heteroatoms, e.g., 1 or1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, independently selected fromnitrogen, oxygen, and sulfur. In the case of bicyclic aromaticheterocyclic or heterocycle or heteroaryl rings, only one of the tworings needs to be aromatic (e.g., 2,3-dihydroindole), though both can be(e.g., quinoline). The second ring can also be fused or bridged asdefined above for heterocycles. The nitrogen atom can be substituted orunsubstituted (i.e., N or NR wherein R is H or another substituent, asdefined). The nitrogen and sulfur heteroatoms can optionally be oxidized(i.e., N→O and S(O)_(p), wherein p = 1 or 2). In certain compounds, thetotal number of S and O atoms in the aromatic heterocycle is not morethan 1.

Examples of aromatic heterocycles, aromatic heterocyclics or heteroarylsinclude, but are not limited to, acridinyl, azocinyl, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, benzooxadiazoly,carbazolyl, 4aH-carbazolyl, carbolinyl, cinnolinyl, furazanyl,imidazolyl, imidazolonyl, 1H-indazolyl, indolizinyl, indolyl,3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylbenztriazolyl,methylfuranyl, methylimidazolyl, methylthiazolyl, naphthyridinyl,oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridooxazolyl,pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyridinonyl, pyridyl,pyrimidinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl,4H-quinolizinyl, quinoxalinyl, tetrahydroquinolinyl, tetrazolyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, triazolopyrimidinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,5-triazolyl, and 1,3,4-triazolyl.

The term “hydroxyalkyl” means an alkyl group as defined above, where thealkyl group is substituted with one or more OH groups. Examples ofhydroxyalkyl groups include HO—CH₂—, HO—CH₂—CH₂— and CH₃—CH(OH)—.

The term “cyano” as used herein means a substituent having a carbon atomjoined to a nitrogen atom by a triple bond, i.e., C═N.

As used herein, “oxo” is means a “═O” group.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds or tautomers thereof, or salts thereof, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds or tautomers thereof, wherein the parentcompound or a tautomer thereof, is modified by making of the acid orbase salts thereof of the parent compound or a tautomer thereof.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound, or a tautomer thereof, formed, for example, fromnon-toxic inorganic or organic acids. For example, such conventionalnon-toxic salts include, but are not limited to, those derived frominorganic and organic acids selected from 2-acetoxybenzoic,2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic,bicarbonic, carbonic, citric, edetic, ethane disulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodide, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic,succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic.

The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound or a tautomer thereof that containsa basic or acidic moiety by conventional chemical methods. Generally,such pharmaceutically acceptable salts can be prepared by reacting thefree acid or base forms of these compounds or tautomers thereof with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington’sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA,USA, p. 1445 (1990).

As used herein, “stable compound” and “stable structure” are meant toindicate a compound that is sufficiently robust to survive isolation toa useful degree of purity from a reaction mixture, and formulation intoan efficacious therapeutic agent.

As used herein, the term “treating” refers to administering a compoundor pharmaceutical composition as provided herein for therapeuticpurposes. The term “therapeutic treatment” refers to administeringtreatment to a patient already suffering from a disease thus causing atherapeutically beneficial effect, such as ameliorating existingsymptoms, ameliorating the underlying metabolic causes of symptoms,postponing or preventing the further development of a disorder, and/orreducing the severity of symptoms that will or are expected to develop.

As used herein, “unsaturated” refers to compounds having at least onedegree of unsaturation (e.g., at least one multiple bond) and includespartially and fully unsaturated compounds.

As used herein, the term “effective amount” refers to an amount of acompound or a pharmaceutically acceptable salt of the compound ortautomer (including combinations of compounds and/or tautomers thereof,and/or pharmaceutically acceptable salts of said compound or tautomer)of the present disclosure that is effective when administered alone orin combination as an antimicrobial agent. For example, an effectiveamount refers to an amount of the compound or tautomer thereof, or apharmaceutically acceptable salt said compound or tautomer that ispresent in a composition, a formulation given to a recipient patient orsubject sufficient to elicit biological activity.

In the specification, the singular forms also include the plural, unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. In the case of conflict, the present specificationwill control. As used herein, “mammal” refers to human and non-humanpatients.

As used herein, the term “formulae of the disclosure” or “formulaedisclosed herein” includes one or more of the Formulae: (I), (Ia),(Ia-1), (Ia-2), (Ib-a), (Ib), (Ia-3), (Ic), (Ic-1), (Id), (Ie), (If),(Ig). (Ie-1), (Ih), (Ii), (Ij), and (Ik).

As used herein, the term “compound of the disclosure” or “compounddisclosed herein” includes one or more compounds of the formulae of thedisclosure or a compound explicitly disclosed herein.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present disclosure also consistessentially of, or consist of, the recited components, and that theprocesses of the present disclosure also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions can be conducted simultaneously.

2. Compounds of the Disclosure

The present application provides compounds of Formula I

or pharmaceutically acceptable salts thereof, wherein:

-   A is CH or O;-   B is CH or N;-   D is C or N;-   E is CH or N;-   W is C or N;-   X is C or N;-   Y is C or N;-   Z is C or N;-   R¹ is H, C₁₋₄ alkyl, phenyl, or hetAr¹;-   R² is H;-   R³ is H;-   or R² and R³, together with Y and X, form a 6-membered cycloalkyl    ring;-   R⁷ is H;-   or, optionally, when n is 0, R¹ and R⁷, together with the atoms to    which they are attached, form a 6-membered cycloalkyl ring;-   R⁴ is H, C₁₋₄ alkyl, halogen, CF₃, or phenyl;-   R⁵ is H, C₁₋₄ alkyl, C₄₋₁₀ cycloalkyl, phenyl optionally substituted    with halogen, (C₁₋₃ alkyl)O(C₄₋₆ cycloalkyl), O(C₁₋₄ alkyl)(C₄₋₆    cycloalkyl), S(C₁₋₄ alkyl), S(C₄₋₆ cycloalkyl), (C₁₋₃ alkyl)(C₄₋₉    hetCyc¹), hetAr¹, or O(phenyl) optionally substituted with CN;-   R⁶ is H or C₁₋₄ alkyl;-   hetAr¹ is a 6-membered heteroaryl ring having 1-3 ring nitrogen    atoms optionally substituted with C₁₋₄ alkyl;-   hetCyc¹ is a 6-10-membered bicyclic ring having at least one ring    heteroatom which is nitrogen and at least one of the rings is    aromatic;-   n is 0 or 1;-   m is 0 or 1;-   p is 0 or 1; and

the dashed lines can be single or double bonds.

In some embodiments, A is CH. In some embodiments, A is O.

In some embodiments, B is CH. In some embodiments, B is N.

In some embodiments, A is O and B is N.

In some embodiments, D is C. In some embodiments, D is N.

In some embodiments, E is CH. In some embodiments, E is N.

In some embodiments, W is N. In some embodiments, W is C.

In some embodiments, X is C.

In some embodiments, Y is C. In some embodiments, Y is N.

In some embodiments, Z is N. In some embodiments, Z is C.

In some embodiments, R¹ is H. In some embodiments, R¹ is C₁₋₄ alkyl orhetAr¹. In some embodiments, R¹ is methyl, isopropyl, phenyl, orpyridine.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is Cl, CF₃, methylor phenyl.

In some embodiments, R⁵ is H or phenyl optionally substituted withhalogen. In some embodiments, R⁵ is phenyl substituted with F. In someembodiments, R⁵ is (C₁₋₃ alkyl)O(C₄₋₆ cycloalkyl), O(C₁₋₄ alkyl)(C₄₋₆cycloalkyl), or O(phenyl) optionally substituted with CN. In someembodiments, C₄₋₆ cycloalkyl is cyclopentyl or cyclohexyl. In someembodiments, R⁵ is (C₁₋₃ alkyl)O(cyclopentyl) or O(C₁₋₄ alkyl)(C₄-₆cyclohexyl). In some embodiments, R⁵ is C₁₋₄ alkyl, C₄₋₁₀ cycloalkyl, or(C₁₋₃ alkyl)(C₄₋₉ hetCyc¹). In some embodiments, hetCyc¹ is a 9-memberedbicyclic ring having one or more nitrogen atoms. In some embodiments,hetCyc¹ is isoindoline. In some embodiments, R⁵ is S(C₁₋₄ alkyl) orS(C₄₋₆ cycloalkyl). In some embodiments, C₄₋₆ cycloalkyl is cyclopentylor cyclohexyl. In some embodiments, R⁵ is hetAr¹. In some embodiments,hetAr¹ is pyridine or pyrimidine optionally substituted with C₁₋₄ alkyl.

In some embodiments, R⁶ is H.

In some embodiments, n is 0.

In some embodiments, m is 1.

In some embodiments, p is 0. In some embodiments, p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H, methyl, isopropyl, phenyl, or a 6-membered heteroaryl ring    having 1 ring nitrogen atom;-   R⁴ is H, halogen, methyl, or CF₃;-   R⁵ is H or phenyl;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R⁴ is H;-   R⁵ is (C₁₋₃ alkyl)O(cyclopentyl), O(C₁₋₄ alkyl)(cyclohexyl), C₁₋₄    alkyl, C₄₋₁₀ cycloalkyl, (C₁₋₃ alkyl)(C₄₋₉ hetCyc¹), S(C₁₋₄ alkyl)    S(C₄₋₆ cyclopentyl) or O(phenyl) optionally substituted with CN;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is C;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R⁴ is H;-   R⁵ is phenyl;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 1; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is O;-   B is N;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R⁴ is phenyl;-   R⁵ is H;-   R⁶ is H;-   R⁷ is H;-   m is 0;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is N;-   X is N;-   Y is N;-   Z is N;-   R¹ is C₁₋₄ alkyl;-   R⁴ is H;-   R⁵ is phenyl;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is C₁₋₄ alkyl;-   R² and R³, together with Y and X, form a 6-membered cycloalkyl ring;-   R⁴ is H;-   R⁵ is phenyl;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R² and R³, together with Y and X, form a 6-membered cycloalkyl ring;-   R⁴ is H or methyl;-   R⁵ is pyridine or pyrimidine substituted with C₁₋₄ alkyl (e.g.,    methyl);-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   W is C;-   Xis C;-   Y is N;-   Z is C;-   R³ is H;-   R¹ and R⁷, together with the atoms to which they are attached, form    a 6-membered cycloalkyl ring;-   R⁴ is H;-   R⁵ is phenyl;-   R⁶ is H;-   m is 1;-   n is 0; and-   p is 0.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is N;-   D is C;-   E is CH;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R² and R³, together with Y and X, form a 6-membered cycloalkyl ring;-   R⁴ is H;-   R⁵ is phenyl;-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula I, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH or N;-   D is N;-   E is CH or N;-   W is N;-   Xis C;-   Y is C;-   Z is N;-   R¹ is H;-   R² and R³, together with Y and X, form a 6-membered cycloalkyl ring;-   R⁵ is phenyl optionally substituted with halogen (e.g., fluoro);-   R⁶ is H;-   R⁷ is H;-   m is 1;-   n is 0; and-   p is 1.

In some embodiments, the compound of Formula I is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is any one of thecompounds listed in Table 1, or a pharmaceutically acceptable salt ofthe compound.

TABLE 1 # Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

The present application also provides compounds of Formula II

or a pharmaceutically acceptable salt thereof, wherein:

-   A is CH, N, or S;-   B is CH, N, O, or S;-   D is C or N;-   E is CH or N;-   L is C₁₋₃ alkylene or C(═O);-   W is CH, CH₂, N, or NR^(a);-   X is CH, CH₂, N, or NR^(b);-   Y is CH, CH₂ or O;-   Z is Nor CR²';-   R¹ is H or C₁₋₃ alkyl;-   R² is absent or C₁₋₆ alkylene;-   R^(2') is H or C₁₋₆ alkyl;-   or, when Z is CR^(2'), R² and R^(2'), together with C, can be taken    together to form a C₁₋₆ heterocyclic ring;-   R³ is H, halogen, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, or C₁₋₃ alkoxy;-   R⁴ is H or C₁₋₃ alkyl;-   R^(4') is H or C₁₋₃ alkyl;-   R^(a) is H or C₁₋₃ alkyl;-   R^(b) is C₁₋₃ alkyl;-   m is 0, 1, or 2;-   n is 1 or 2;-   p is 0 or 1; and-   the dashed lines can be single or double bonds;-   with the proviso that when n is 2, R¹ is methyl, D is C, R³ is H,    and A, B, and E are all CH, then at least one of W, X, and Y is not    CH₂.

In some embodiments, A is CH. In some embodiments, A is N. In someembodiments, A is S.

In some embodiments, B is CH. In some embodiments, B is N, O, or S.

In some embodiments, D is C.

In some embodiments, E is CH. In some embodiments, E is N.

In some embodiments, L is C₁₋₃ alkylene. In some embodiments, L ismethylene.

In some embodiments, W is CH₂. In some embodiments, W is N.

In some embodiments, X is CH. In some embodiments, X is CH₂. In someembodiments, X is NR^(b).

In some embodiments, R^(b) is methyl.

In some embodiments, Y is CH. In some embodiments, Y is CH₂.

In some embodiments, Z is N. In some embodiments, Z is CR^(2').

In some embodiments, R² is absent. In some embodiments, R² and R^(2'),together with C, form a 5-membered heterocyclic ring having 1 nitrogenatom.

In some embodiments, R¹ is H. In some embodiments, R¹ is methyl.

In some embodiments, R^(a) is H or methyl.

In some embodiments, R^(b) is methyl.

In some embodiments, R³ is H, halogen, or C₃₋₆ cycloalkyl. In someembodiments, R³ is halogen. In some embodiments, R³ is chlorine. In someembodiments, R³ is cyclopropyl.

In some embodiments, R⁴ and R^(4') are each H. In some embodiments, R⁴and R^(4') are each methyl.

In some embodiments, m is 1 or 2.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, p is 0. In some embodiments, p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is N, O, or S;-   D is C or N;-   E is CH or N;-   L is C₁₋₃ alkylene;-   W is CH₂;-   X is CH₂ or NR^(b);-   Y is CH₂;-   Z is N;-   R¹ is H or C₁₋₃ alkyl;-   R² is absent;-   R³ is C₁₋₃ alkyl;-   R⁴ is H;-   R^(4') is H;-   R^(b) is C₁₋₃ alkyl;-   n is 2;-   m is 1; and-   p is 0.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is CH₂;-   X is NR^(b);-   Y is CH₂;-   Z is N;-   R¹ is H;-   R² is absent;-   R³ is C₁₋₃ alkoxy;-   R⁴ is H;-   R^(4') is H;-   R^(b) is C₁₋₃ alkyl;-   n is 2;-   m is 1; and-   p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is NR^(a);-   X is N;-   Y is CH;-   Z is N;-   R¹ is H or C₁₋₃ alkyl;-   R² is absent;-   R³ is H;-   R⁴ is H;-   R^(4') is H;-   R^(a) is C₁₋₃ alkyl;-   n is 2;-   m is 0; and-   p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is NR^(a);-   X is CH₂;-   Y is O;-   Z is N;-   R¹ is H;-   R² is absent;-   R³ is H;-   R⁴ is H;-   R^(4') is H;-   R^(a) is H;-   n is 2;-   m is 1; and-   p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C(═O) or C₁₋₃ alkylene;-   W is CH₂;-   X is CH₂;-   Y is CH₂;-   Z is N;-   R¹ is C₁₋₃ alkyl;-   R² is absent;-   R³ is H;-   R⁴ is H or C₁₋₃ alkyl;-   R^(4') is H or C₁₋₃ alkyl;-   n is 1;-   m is 1; and-   p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is CH₂;-   X is CH₂;-   Y is CH₂;-   Z is CR^(2'), and R² and R^(2'), together with C, are taken together    to form a C₅₋₆ heterocyclic ring;-   R¹ is C₁₋₃ alkyl;-   R³ is H;-   R⁴ is H;-   R^(4') is H;-   n is 1;-   m is 1; and-   p is 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH or S;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is CH₂;-   X is CH₂;-   Y is CH₂;-   Z is N;-   R¹ is methyl;-   R² is absent;-   R³ is H or halogen (e.g., chloro);-   R⁴ is H;-   R^(4') is H;-   m is 1;-   n is 2; and-   p is 0 or 1.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is N;-   B is O;-   D is C;-   E is N;-   L is C₁₋₃ alkylene;-   W is CH₂;-   X is CH₂;-   Y is CH₂;-   Z is N;-   R¹ is methyl;-   R² is absent;-   R³ is C₃₋₆ cycloalkyl;-   R⁴ is H;-   R^(4') is H;-   m is 1;-   n is 2; and-   p is 0.

In some embodiments, provided is a compound of Formula II, or apharmaceutically acceptable salt thereof, wherein:

-   A is CH;-   B is CH;-   D is C;-   E is CH;-   L is C₁₋₃ alkylene;-   W is N;-   X is CH;-   Y is CH;-   Z is N;-   R¹ is H;-   R² is absent;-   R³ is H;-   R⁴ is H;-   R^(4') is H;-   m is 1;-   n is 2; and-   p is 1.

In some embodiments, the compound of Formula II is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula II is a compound of FormulaIIa:

or a pharmaceutically acceptable salt thereof, wherein:

-   A is CH or N;-   B is N, O, or S;-   D is C or N;-   E is CH or N;-   W is CH₂ or NR^(a);-   X is CH, CH₂, N, or NR^(b);-   Y is CH, CH₂ or O;-   L is C₁₋₃ alkylene or C(═O);-   R¹ is H or C₁₋₃ alkyl;-   R³ is C₁₋₃ alkyl or C₁₋₃ alkoxy;-   R⁴ is H or C₁₋₃ alkyl;-   R^(4') is H or C₁₋₃ alkyl;-   R^(a) is H or C₁₋₃ alkyl;-   R^(b) is C₁₋₃ alkyl;-   n is 1 or 2;-   m is 0, 1 or 2;-   p is 0 or 1; and-   the dashed lines can be single or double bonds.

In some embodiments, the compound of Formula II is a compound of FormulaIIb:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is C₁₋₃ alkyl; and-   R⁵ is (C₁₋₃ alkyl)phenyl.

In some embodiments, the compound of Formula II is any one of thecompounds listed in Table 2, or a pharmaceutically acceptable salt ofthe compound.

TABLE 2 # Structure 31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

3. Synthesis of the Compounds of the Disclosure

As will be appreciated, the compounds provided herein, including saltsthereof, can be prepared using known organic synthesis techniques andcan be synthesized according to any of numerous possible syntheticroutes. The compounds thus obtained can be further purified, forexample, by flash column chromatography, high performance liquidchromatography, crystallization, or any known purification method.

In one embodiment, compounds of the present disclosure, e.g., compoundsof Formula I and Formula II can be synthesized according to theprocedures illustrated in synthetic Schemes 1-4 below.

Compounds of Formula I having an imidazopyridine structure can beprepared, for example, using the generalized processes illustrated inSchemes 1 and 2. The proposed synthetic route has three key stages -formation of substituted imidazo[1,2-a]pyridine-2-carbaldehydes (stepA), acylation of anilines (step B), and synthesis of final compounds(step C). Step A is well documented in literature (see, e.g., Chavignonet al. (1992) J. Heterocycl. Chem. 29(4):691) and consists of twostages: 1) formation of corresponding2-(dichloromethyl)imidazo[1,2-a]pyridines by condensation of substituted2-aminopyridines with 1,1,3-trichloroacetone in 1,2-dimethoxyethane atheating; 2) transformation of 2-(dichloromethyl)imidazo[1,2-a]pyridinesat their treatment by calcium carbonate in correspondingimidazo[1,2-a]pyridine-2-carbaldehydes. Step B also has two stages -synthesis of amides of boc-protected aminoacetic acid (R3 = H) to avoidformation of side products and the removal of protecting boc-group underacidic conditions. Final step C implies in formation of Schiff bases atthe reaction of imidazo[1,2-a]pyridine-2-carbaldehydes withcorresponding amines followed by their reduction by sodium borohydride.

In cases when R3 = alkane, the synthetic route is shorter and involves 3stages -alkylation of Alk-substituted aminoacetic acid tert-butyl estersby corresponding 2-(chloromethyl)imidazo[1,2-a]pyridines (step D, Scheme2), hydrolysis (step E, Scheme 2), and final amides formation (step F,Scheme 2).

Compounds of Formula I having an imidazole structure can be prepared,for example, using the generalized process illustrated in Scheme 3. Theprocess includes amide bond formation by reaction of correspondingimidazolyl-acetic acids with anilines. Depending on the chemical natureof R1 and R2, different activating agents such as carbodiimides (DCC,EDC), carbonyl diimidazole (CDI) may be used (see, e.g., Montalbetti etal. (2005) Tetrahedron 61:10827).

Compounds of Formula II having a pyrimidine/homopiperazine scaffold canbe prepared, for example, using the generalized process illustrated inScheme 4.

It will be appreciated by one skilled in the art that the processesdescribed herein are not the exclusive means by which compounds providedherein may be synthesized and that a broad repertoire of syntheticorganic reactions is available to be potentially employed insynthesizing compounds provided herein. The person skilled in the artknows how to select and implement appropriate synthetic routes. Suitablesynthetic methods of starting materials, intermediates and products maybe identified by reference to the literature, including referencesources such as: Advances in Heterocyclic Chemistry, Vols. 1-107(Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49(Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.)Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge UpdatesKU2010/1-4; 2011/1-4; 2012/1-2 (Thieme, 2001-2012); Katritzky, et al.(Ed.) Comprehensive Organic Functional Group Transformations, (PergamonPress, 1996); Katritzky et al. (Ed.); Comprehensive Organic FunctionalGroup Transformations II (Elsevier, 2^(nd) Edition, 2004); Katritzky etal. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984);Katritzky et al., Comprehensive Heterocyclic Chemistry II, (PergamonPress, 1996); Smith et al., March’s Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley, 2007); Trost etal. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

The reactions for preparing compounds described herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,(e.g., temperatures which can range from the solvent’s freezingtemperature to the solvent’s boiling temperature). A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds described herein can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC). Compounds can be purified bythose skilled in the art by a variety of methods, including highperformance liquid chromatography (HPLC) and normal phase silicachromatography.

Compounds provided herein also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone - enol pairs, amide -imidic acidpairs, lactam - lactim pairs, enamine - imine pairs, and annular formswhere a proton can occupy two or more positions of a heterocyclicsystem, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates) or can be isolated.

In some embodiments, preparation of compounds can involve the additionof acids or bases to affect, for example, catalysis of a desiredreaction or formation of salt forms such as acid addition salts.

Example acids can be inorganic or organic acids and include, but are notlimited to, strong and weak acids. Some example acids includehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid,benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weakacids include, but are not limited to acetic acid, propionic acid,butanoic acid, benzoic acid, tartaric acid, pyroglutamic acid, gulonicacid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid,nonanoic acid, and decanoic acid. Also included are organic diacids suchas malonic, fumaric and maleic acid.

Example bases include lithium hydroxide, sodium hydroxide, potassiumhydroxide, lithium carbonate, sodium carbonate, potassium carbonate, andsodium bicarbonate. Some example strong bases include, but are notlimited to, hydroxide, alkoxides, metal amides, metal hydrides, metaldialkylamides and arylamines, wherein; alkoxides include lithium, sodiumand potassium salts of methyl, ethyl and t-butyl oxides; metal amidesinclude sodium amide, potassium amide and lithium amide; metal hydridesinclude sodium hydride, potassium hydride and lithium hydride; and metaldialkylamides include lithium, sodium, and potassium salts of methyl,ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl andcyclohexyl substituted amides.

In some embodiments, the compounds provided herein, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

4. Methods of Use

Provided herein are methods of activating the enzymatic activity an E3ubiquitin ligase in a subject in need thereof. As used herein, the term“subject,” refers to any animal, including mammals. For example, mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,primates, and humans. In some embodiments, the subject is a human. Insome embodiments, the method comprises administering to the subject atherapeutically effective amount of a compound provided herein (e.g., acompound of Formula I or Formula II, or a pharmaceutically acceptablesalt thereof).

In some embodiments, the subject has diminished E3 ubiquitin ligaseenzymatic activity. Examples of E3 ubiquitin ligases include, but arenot limited to, Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31 (HOIP),RBCK1 (HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2. In someembodiments, the E3 ubiquitin ligase is Parkin.

In some embodiments of the methods provided herein, the enzymaticactivity is activated or enhanced during mitochondrial stress. In someembodiments, the compound, e.g., a compound of Formula I or Formula II,or a pharmaceutically acceptable salt thereof, stimulates mitochondrialquality control. In some embodiments, the compound, e.g., a compound ofFormula I or Formula II, or a pharmaceutically acceptable salt thereof,interferes with the auto-inhibition of the ligase.

In some embodiments, the diminished E3 ubiquitin ligase enzymaticactivity is due to a disease, aging, or an age-related disorder.Examples of diseases or disorders include, but are not limited to,Parkinson’s disease, parkinsonism, Alzheimer’s disease, dementia,Amyotrophic lateral sclerosis, Frontotemporal dementia, autism,depression, leprosy, an inclusion body myositis, diabetes mellitus,diabetic kidney disease, a liver disease, a lysosomal storage disorder,a neurological disease, a muscular disease, a mitochondrial disease, andcancer.

In some embodiments, the disease or disorder is Parkinson’s disease.

In some embodiments, the disease or disorder is cancer. Example ofcancers include, but are not limited to, liver cancer, brain cancer,skin cancer, kidney cancer, lung cancer, colon cancer, pancreaticcancer, hepatocellular carcinoma, glioma, skin cutaneous melanoma, clearcell renal cell carcinoma, non-small cell lung cancer, lungadenocarcinoma, lung squamous cell cancer, colorectal cancer, pancreaticadenocarcinoma, adenoid cystic carcinoma, acute lymphoblastic leukemia,chronic myeloid leukemia, bladder urothelial cancer, head and necksquamous cell carcinoma, esophageal adenocarcinoma, gastric cancer,cervical cancer, thyroid cancer, and endometrioid cancer.

The present application further provides a method treating a disease ordisorder associated with diminished E3 ubiquitin ligase enzymaticactivity in a subject. In some embodiments, the subject is a human. Insome embodiments, the method comprises administering to the subject atherapeutically effective amount of a compound provided herein, e.g., acompound of Formula I or Formula II, or a pharmaceutically acceptablesalt thereof.

Provided herein is a method of treating a disease or disorder in asubject. In some embodiments, the subject is a human. In someembodiments, the method comprises:

-   (a) determining if the disease or disorder is associated with    diminished E3 ubiquitin ligase enzymatic activity; and-   (b) if the disease is determined to be associated with diminished E3    ubiquitin ligase enzymatic activity, administering to the subject a    therapeutically effective amount of a compound provided herein,    e.g., a compound of Formula I or Formula II, or a pharmaceutically    acceptable salt thereof.

In some embodiments, the method of treating a disease or disorder in asubject comprises:

-   (a) detecting a disease or disorder associated with diminished E3    ubiquitin ligase enzymatic activity; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, e.g., a compound of Formula I or    Formula II, or a pharmaceutically acceptable salt thereof.

Provided herein is a method of treating Parkinson’s disease in asubject. In some embodiments, the subject is a human. In someembodiments, the method comprises:

-   (a) determining if the subject has Parkinson’s disease; and-   (b) if the subject has Parkinson’s disease, administering to the    subject a therapeutically effective amount of a compound provided    herein, e.g., a compound of Formula I or Formula II, or a    pharmaceutically acceptable salt thereof.

In some embodiments, the method of treating Parkinson’s disease in asubject comprises:

-   (a) detecting Parkinson’s disease in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, e.g., a compound of Formula I or    Formula II, or a pharmaceutically acceptable salt thereof.

Provided herein is a method of treating an age-related disorder in asubject. In some embodiments, the subject is a human. In someembodiments, the method comprises:

-   (a) determining if the subject has an age-related disorder; and-   (b) if the subject has an age-related disorder, administering to the    subject a therapeutically effective amount of a compound provided    herein, e.g., a compound of Formula I or Formula II, or a    pharmaceutically acceptable salt thereof.

In some embodiments, the method of treating an age-related disorder in asubject comprises:

-   (a) detecting an age-related disorder in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, e.g., a compound of Formula I or    Formula II, or a pharmaceutically acceptable salt thereof.

Provided herein is a method of treating cancer in a subject. In someembodiments, the subject is a human. In some embodiments, the methodcomprises:

-   (a) determining if the subject has cancer; and-   (b) if the subject has cancer, administering to the subject a    therapeutically effective amount of a compound provided herein,    e.g., a compound of Formula I or Formula II, or a pharmaceutically    acceptable salt thereof.

In some embodiments, the method of treating cancer in a subjectcomprises:

-   (a) detecting cancer in a subject; and-   (b) administering to the subject a therapeutically effective amount    of a compound provided herein, e.g., a compound of Formula I or    Formula II, or a pharmaceutically acceptable salt thereof.

Example of cancers include, but are not limited to, liver cancer, braincancer, skin cancer, kidney cancer, lung cancer, colon cancer,pancreatic cancer, hepatocellular carcinoma, glioma, skin cutaneousmelanoma, clear cell renal cell carcinoma, non-small cell lung cancer,lung adenocarcinoma, lung squamous cell cancer, colorectal cancer,pancreatic adenocarcinoma, adenoid cystic carcinoma, acute lymphoblasticleukemia, chronic myeloid leukemia, bladder urothelial cancer, head andneck squamous cell carcinoma, esophageal adenocarcinoma, gastric cancer,cervical cancer, thyroid cancer, and endometrioid cancer.

Provided herein are methods of activating the enzymatic activity of anE3 ubiquitin ligase in a cell. In some embodiments, the method comprisescontacting the cell with a compound provided herein, e.g., a compound ofFormula I or Formula II, or a pharmaceutically acceptable salt thereof.In some embodiments, the contacting is in vitro.

In some embodiments of the methods provided herein, the enzymaticactivity is activated or enhanced during mitochondrial stress. In someembodiments, the compound, e.g., a compound of Formula I or Formula II,or a pharmaceutically acceptable salt thereof, stimulates mitochondrialquality control. In some embodiments, the compound, e.g., a compound ofFormula I or Formula II, or a pharmaceutically acceptable salt thereof,interferes with the auto-inhibition of the ligase. In some embodiments,the cell has diminished E3 ubiquitin ligase enzymatic activity.

In some embodiments of any of the methods provided herein, the compound(e.g., a compound of Formula I or Formula II) for use in the methodsdescribed herein may be used in combination with one or more of thecompounds provided and described in the present disclosure.

5. Combination Therapies

In some embodiments, one or more of the compounds provided herein can beadministered to a subject in need thereof in combination with at leastone additional pharmaceutical agent. In some embodiments, the additionalpharmaceutical agent is a compound provided herein (e.g., a compound ofFormula I or Formula II).

Additional examples of suitable additional pharmaceutical agents for usein combination with the compounds of the present application fortreatment of the diseases or disorders provided herein include, but arenot limited to, activators of general autophagy, such as rapamycin andresveratrol (e.g., mTOR pathway inhibition) or AMPK pathway activation(e.g., metformin); activators of lysosomal function/biogenesis, e.g.,stimulation of the master regulator TFEB; activators of PINK1 kinaseactivity, e.g., kinetin; and molecules that activate Parkin byalternative mechanisms, such as phosphor-ubiquitin binding, includingphosphor-ubiquitin mimics. In some embodiments, the compounds providedherein may be administered to a subject in need thereof in combinationwith at least one additional pharmaceutical agent for the treatment of adisease or disorder associated with decreased or diminished E3 ubiquitinligase activity. In some embodiments, the enzymatic activity isdiminished due to a disease, aging, or an age-related disorder.

6. Pharmaceutical Compositions and Formulations

Provided herein are pharmaceutical compositions comprising a compoundprovided herein (e.g., a compound of Formula I or Formula II, or apharmaceutically acceptable salt thereof), and at least onepharmaceutically acceptable carrier. When employed as pharmaceuticals,the compounds provided herein can be administered in the form ofpharmaceutical compositions; thus, the methods described herein caninclude administering the pharmaceutical compositions. Thesecompositions can be prepared as described herein or elsewhere, and canbe administered by a variety of routes, depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal orintranasal), oral, or parenteral. Parenteral administration may include,but is not limited to intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular injection or infusion; or intracranial,(e.g., intrathecal, intraocular, or intraventricular) administration.Parenteral administration can be in the form of a single bolus dose, ormay be, for example, by a continuous perfusion pump. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. In some embodiments, thecompounds provided herein are suitable for oral and parenteraladministration. In some embodiments, the compounds provided herein aresuitable for oral administration. In some embodiments, the compoundsprovided herein are suitable for parenteral administration. In someembodiments, the compounds provided herein are suitable for intravenousadministration. In some embodiments, the compounds provided herein aresuitable for transdermal administration (e.g., administration using apatch or microneedle). Pharmaceutical compositions for topicaladministration may include transdermal patches (e.g., normal orelectrostimulated), ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

Also provided are pharmaceutical compositions which contain, as theactive ingredient, a compound provided herein (e.g., a compound ofFormula I or Formula II), or a pharmaceutically acceptable salt thereof,in combination with one or more pharmaceutically acceptable carriers(excipients). In making the compositions provided herein, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

Some examples of suitable excipients include, without limitation,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, and methyl cellulose. The formulations can additionally include,without limitation, lubricating agents such as talc, magnesium stearate,and mineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; flavoring agents, or combinations thereof.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered and the schedule of administration will usually bedetermined by a physician, according to the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound administered, the age, weight, andresponse of the individual subject, the severity of the subject’ssymptoms, and the like.

7. Kits

Also provided herein are kits including a compound provided herein, moreparticularly, a compound of Formula I or Formula II, or apharmaceutically acceptable salt thereof. In some embodiments, a kit caninclude one or more delivery systems, e.g., for a compound providedherein, or a pharmaceutically acceptable salt thereof, and directionsfor use of the kit (e.g., instructions for treating a subject). In someembodiments, a kit can include a compound provided herein, or apharmaceutically acceptable salt thereof, and one or more additionalagents as provided herein.

In some embodiments, the compound is selected from the group ofcompounds provided in Table 1, or a pharmaceutically acceptable saltthereof. In some embodiments, the compound is selected from the group ofcompounds provided in Table 2, or a pharmaceutically acceptable saltthereof.

In some embodiments, the kit can include one or more compounds oradditional pharmaceutical agents as provided herein, or apharmaceutically acceptable salt thereof, and a label that indicatesthat the contents are to be administered to a subject suffering for adisease or disorder associated with diminished or decreased E3 ubiquitinligase enzymatic activity. In some embodiments, a kit can include one ormore compounds as provided herein, or a pharmaceutically acceptable saltthereof, and a label that indicates that the contents are to beadministered with one or more additional pharmaceutical agents asprovided herein.

EXAMPLES Example 1: Synthetic Routes

Compounds 1-46, listed in Table 3 below, were synthesized according tothe general synthetic schemes described below.

TABLE 3 Compounds 1-46 # Structure EC₅₀ (nM) 1

371 2

549 3

405 4

164 5

63 6

2000 7

2000 8

5000 9

5000 10

5000 11

5000 12

5000 13

5000 14

5000 15

5000 16

5000 17

5000 18

5000 19

10000 20

10000 21

238 22

246 23

272 24

190 25

382 26

938 27

190 28

134 29

2000 30

5000 31

990 32

36 33

111 34

NA 35

10000 36

>10000 37

NA 38

NA 39

NA 40

>10000 41

NA 42

707 43

280 44

438 45

366 46

203

A. Synthesis of Compounds 1, 3, 8-10, 12, and 15-20

Compounds 1, 3, 8-10, 12, and 15-20 can be prepared according to thegeneral synthetic scheme below:

Experimental:

A mixture of (3-{[(ethylimino)methylidene]amino}propyl)dimethylaminehydrochloride (EDC-HCl) (1.2 mmol), 1-hydroxybenzotriazole (HOBt) (1.2mmol), N,N-diisopropylethylamine (0.35 mL, 2 mmol), acid (1.2 mmol), andaniline (1 mmol) in dimethylformamide (7 mL) was stirred for 24-48 hoursat room temperature. Then the reaction mixture was treated with water(35 mL), the crude product was filtered and purified byrecrystallization from acetonitrile or by HPLC chromatography(methanol/water). Yield: 15-70% depending on the structures of acid andaniline.

The spectral data for each of Compounds 1, 3, 8-10, 12, and 15-20 islisted below:

-   Compound 1:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 7.7 (m, 6H, C_(Ph)H),        7.4 (m, 3H, C_(Ph)H), 7.35 (s, 1H, C_(imid)H), 4.85 (s, 2H,        CH₂), 2.4 (t, 4H, 2CH₂), 1.75 (m, 4H, 2CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 332.2/332.2.-   Compound 3:    -   ¹H NMR (DMSO-d₆): δ = 10.45 (s, 1H, NH), 7.6 (m, 3H, 2C_(Ph)H,        C_(imid)H), 7.45 (dd, 2H, C_(Ph)H), 7.16 (d, 1H, C_(imid)H), 6.9        (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂), 1.2 (s, 9H, 3CH₃) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 290.15/290.2.-   Compound 8:    -   ¹H NMR (DMSO-d₆): δ = 10.2 (s, 1H, NH), 7.65 (s, 1H, C_(imid)H),        7.45 (dd, 2H, C_(Ph)H), 7.15 (d, 1H, C_(imid)H), 6.9 (m, 3H,        2C_(Ph)H, C_(imid)H), 4.85 (s, 2H, CH₂), 3.75 (d, 2H, OCH₂),        1.75 (m, 6H, 3CH₂), 1.2 (m, 3H, CH, CH₂), 1.05 (m, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 314.21/314.1.-   Compound 9:    -   ¹H NMR (DMSO-d₆): δ = 10.35 (s, 1H, NH), 7.65 (s, 1H,        C_(imid)H), 7.56 (dd, 2H, C_(Ph)H), 7.32 (dd, 2H, C_(Ph)H), 7.18        (d, 1H, C_(imid)H), 7.04 (d, 1H, C_(Ph)H), 6.98 (t, 1H,        C_(Ph)H), 6.9 (d, 1H, C_(imid)H), 6.58 (m, 2H, C_(Ph)H), 4.8 (s,        2H, CH₂), 4.2 (s, 2H, NCH₂), 3.25 (t, 2H, CH₂), 2.9 (t, 2H, CH₂)        ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 333.19/333.1.-   Compound 10:    -   ¹H NMR (DMSO-d₆): δ = 10.25 (s, 1H, NH), 7.65 (s, 1H,        C_(imid)H), 7.45 (dd, 2H, C_(Ph)H), 7.3 (dd, 2H, C_(Ph)H), 7.1        (d, 1H, C_(imid)H), 6.85 (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂),        2.05 (m, 3H, C_(adam)H), 1.8 (m, 12H, C_(adam)H₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 336.24/336.1.-   Compound 12:    -   ¹H NMR (DMSO-d₆): δ = 10.8 (s, 1H, NH), 8.2 (d, 1H, C_(Ph)H),        7.85 (m, 1H, C_(Ph)H), 7.65 (s, 1H, C_(imid)H), 7.4 (m, 4H,        C_(Ph)H), 7.3 (m, 2H, C_(Ph)H), 7.2 (d, 1H, C_(imid)H), 6.9 (d,        1H, C_(imid)H), 5.0 (s, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 346.3/346.2.-   Compound 15:    -   ¹H NMR (DMSO-d₆): δ = 10.35 (s, 1H, NH), 7.65 (s, 1H,        C_(imid)H), 7.56 (dd, 2H, C_(Ph)H), 7.26 (dd, 2H, C_(Ph)H), 7.16        (d, 1H, C_(imid)H), 6.9 (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂),        4.4 (s, 2H, OCH₂), 3.9 (m, 1H, CH), 1.65 (m, 6H, CH₂), 1.5 (m,        2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 300.19/300.2.-   Compound 16:    -   ¹H NMR (DMSO-d₆): δ = 10.55 (s, 1H, NH), 7.9 (dd, 2H, C_(Ph)H),        7.7 (dd, 2H, C_(Ph)H), 7.65 (s, 1H, C_(imid)H), 7.1 (m, 5H,        4C_(Ph)H, C_(imid)H), 6.9 (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂)        ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 319.12/319.2.-   Compound 17:    -   ¹H NMR (DMSO-d₆): δ = 10.2 (s, 1H, NH), 7.65 (s, 1H, C_(imid)H),        7.45 (dd, 2H, C_(Ph)H), 7.3 (d, 1H, C_(imid)H), 7.2 (dd, 2H,        C_(Ph)H), 6.9 (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂), 2.4 (d, 2H,        CH2), 1.8 (m, 1H, C_(isoprop)H), 0.9 (d, 6H, 2C_(isoprop)H3)        ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 258.18/258.2.-   Compound 18:    -   ¹H NMR (DMSO-d₆): δ = 10.35 (s, 1H, NH), 7.65 (s, 1H,        C_(imid)H), 7.56 (dd, 2H, C_(Ph)H), 7.34 (dd, 2H, C_(Ph)H), 7.18        (d, 1H, C_(imid)H), 6.9 (d, 1H, C_(imid)H), 4.9 (s, 2H, CH₂),        3.55 (quint, 1H, CH), 2 (m, 2H, CH₂), 1.7 (m, 2H, CH₂), 1.5 (m,        4H, 2CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 302.15/302.2.-   Compound 19:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 7.7 (m, 6H, C_(Ph)H),        7.4 (m, 3H, C_(Ph)H), 7.1 (d, 1H, C_(imid)H), 6.75 (d, 1H,        C_(imid)H), 4.85 (s, 2H, CH₂), 2.3 (s, 3H, CH₃) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 292.16/292.2.-   Compound 20:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 8.55 (d, 2H, C_(pyr)H),        7.7 (m, 6H, C_(Ph)H), 7.4 (m, 5H, 3C_(Ph)H, 2C_(pyr)H), 3.8 (s,        2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 289.15/289.0.

B. Synthesis of Compounds 2, 4, 6, 7, 11, and 14

Compounds 2, 4, 6, 7, 11, and 14 may be prepared according to thegeneral synthetic scheme below:

Experimental:

A mixture of corresponding 2-chloro-N-phenylacetamide (1 mmol),imidazole (1.5 mmol), N,N-diisopropylethylamine (0.35 ml, 2 mmol) indimethylformamide (7 mL) was stirred for 16 hours at 60° C. Then thereaction mixture was cooled to room temperature and treated with water(35 ml). The crude product was filtered and purified byrecrystallization from acetonitrile or by HPLC chromatography(methanol/water). Yield: 15-70% depending on the structures of thestarting materials.

The spectral data for each of Compounds 2, 4, 6, 7, 11, and 14 is listedbelow:

-   Compound 2:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 7.7 (m, 6H, C_(Ph)H),        7.5 (m, 2H, C_(Ph)H), 7.35 (m, 1H, C_(Ph)H), 7.1 (d, 1H,        C_(imid)H), 6.8 (d, 1H, C_(imid)H), 4.95 (s, 2H, CH₂), 3.0 (sep,        1H, C_(isoprop)H), 1.2 (d, 6H, 2C_(isoprop)H₃) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 320.2/320.2.-   Compound 4:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 8.5 (d, 1H, C_(pyr)H),        8.15 (d, 1H, C_(pyr)H), 7.9 (t, 1H, C_(pyr)H), 7.65 (m, 6H,        C_(Ph)H), 7.45 (m, 2H, C_(Ph)H), 7.4 (d, 1H, C_(imid)H), 7.35        (m, 2H, C_(Ph)H, C_(pyr)H), 7.1 (d, 1H, C_(imid)H), 5.5 (s, 2H,        CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 355.17/355.2.-   Compound 6:    -   ¹H NMR (DMSO-d₆): δ = 10.55 (s, 1H, NH), 7.9 (t, 1H, C_(Ph)H),        7.7 (d, 1H, C_(Ph)H), 7.65 (s, 1H, C_(imid)H), 7.45 (m, 6H,        C_(Ph)H), 7.2 (d, 1H, C_(imid)H), 6.9 (d, 1H, C_(imid)H), 5.0        (s, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 312.1/312.2.-   Compound 7:    -   ¹H NMR (DMSO-d₆)_(:) δ = 10.45 (s, 1H, NH), 7.66 (m, 6H,        C_(ph)H), 7.62 (m, 2H, C_(ph)H), 7.46 (m, 5H, C_(ph)H), 7.35 (m,        2H, C_(ph)H, C_(imid)H), 7.1 (d, 1H, C_(imid)H), 4.95 (s, 2H,        CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 354.18/354.1.-   Compound 11:    -   ¹H NMR (DMSO-d₆): δ = 10.5 (s, 1H, NH), 8.85 (d, 1H, C_(pyr)H),        8.6 (d, 1H, C_(pyr)H), 8.05 (d, 1H, C_(pyr)H), 7.65 (m, 6H,        C_(ph)H), 7.45 (m, 3H, 2C_(ph)H_(,)C_(pyr)H), 7.4 (d, 1H,        C_(imid)H), 7.3 (m, 1H, C_(ph)H), 7.1 (d, 1H, C_(imid)H), 5.05        (s, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 355.17/355.2.-   Compound 14:    -   ¹H NMR (DMSO-d₆): δ = 10.55 (s, 1H, NH), 7.62 (m, 2H, C_(ph)H,        C_(imid)H), 7.57 (m, 1H, C_(ph)H), 7.35 (t, 2H, C_(ph)H), 7.06        (m, 2H, C_(ph)H, C_(imid)H), 6.95 (m, 4H, C_(ph)H), 6.86 (d, 1H,        C_(imid)H), 4.85 (s, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 294.13/294.2.

C. Synthesis of Compound 30

Compound 30 may be prepared according to the synthetic scheme below:

Experimental:

A mixture of (3-{[(ethylimino)methylidene]amino}propyl)dimethylaminehydrochloride (EDC-HCI) (1.2 mmol), 1-hydroxybenzotriazole (HOBt) (1.2mmol), N,N-diisopropylethylamine (0.35 ml, 2 mmol),2-(1H-imidazol-1-yl)acetic acid (1.2 mmol), and 3-methyl-4-phenylaniline(1 mmol) in dimethylformamide (7 mL) was stirred for 36 hours at roomtemperature. Then the reaction mixture was treated with water (35 mL),the crude product was filtered and purified by recrystallization fromacetonitrile. Yield: 40%.

The spectral data for Compound 30 is listed below:

-   Compound 30:    -   ¹H NMR (DMSO-d₆): δ = 10.55 (s, 1H, NH), 7.65 (s, 1H,        C_(imid)H),, 7.45 (m, 4H, C_(ph)H), 7.37 (m, 3H, C_(ph)H) ,7.17        (m, 2H, C_(ph)H, C_(imid)H), 6.9 (d, 1H, C_(imid)H), 4.9 (s, 2H,        CH₂), 2.2 (s, 3H, CH₃) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 292.16.1 292.0.

D. Synthesis of Compound 5

Compound 5 may be prepared according to the synthetic scheme below:

The spectral data from compound 5 is listed below:

-   Compound 5:    -   ¹H NMR (DMSO-d₆): δ = 10.9 (s, 1H, NH), 7.95 (d, 1H, C_(py)H),        7.65 (d, 1H, C_(py)H), 7.3-7.5 (m, 6H, C_(ph)H; C_(imid)H), 4.85        (s, 2H, CH₂CO), 2.4 (m, 4H, 2CH₂; s, 3H, CH₃), 1.7 (m, 4H, 2CH₂)        ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 347.21/347.2.

E. Synthesis of Compound 31

A mixture of 4-chloro-2-methyl-5,6,7,8-tetrahydroquinazoline (0.183 g, 1mmol), 1-benzyl-1,4-diazepan (0.228 g, 1.2 mmol), andN,N-diisopropylethylamine (0.35 mL, 2 mmol) in dimethylacetamide (7 mL)was stirred for 16 hours at 90° C. Then the reaction mixture was cooledto room temperature and treated with brine (35 mL). The mixture wasextracted with ethyl acetate (2 × 70 mL). The combined organic layerswere washed with water (70 mL), brine (70 mL), dried over Na₂SO₄,filtered, and concentrated under reduced pressure. Purification by HPLCchromatography (methanol/water) afforded 0.188 g (0.56 mmol, 56% yield)of Compound 31 as an oil. LS-MS (m/z): calcd./found for [M+H]⁺337.48/337.4.

F. Synthesis of Compound 32

Compound 32 may be prepared according to the synthetic scheme below:

The spectral data from compound 32 is listed below.

-   Compound 32:    -   ¹H NMR (DMSO-d₆): δ = 8.3 (s, 1H, C_(quin)H), 7.3 (m, 5H,        C_(ph)H), 3.55 (s, 2H, CH₂Ph), 3.5 (m, 4H, 2CH₂), 2.7 (m, 4H,        2CH₂), 2.55 (m, 4H, 2CH₂), 1.85 (m, 2H, CH₂), 1.75 (m, 2H, CH₂),        1.6 (m, 4H, 2CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 337.28/337.2.

G. Synthesis of Compound 33

Compound 33 may be prepared according to the synthetic scheme below:

The spectral data from compound 33 is listed below.

-   Compound 33:    -   ¹H NMR (DMSO-d₆): δ = 8.3 (s, 1H, C_(quin)H), 7.3 (m, 5H,        C_(ph)H), 3.65 (m, 4H, 2CH₂), 3.55 (s, 2H, CH₂Ph), 2.7 (m, 4H,        2CH₂), 2.55 (m, 4H, 2CH₂), 1.85 (m, 2H, CH₂), 1.75 (m, 2H, CH₂),        1.6 (m, 2H, CH₂) ppm.    -   LS-MS (m/z): calcd./found for [M+H]⁺ 323.26/323.2.

Example 2: Ames Test of Compounds 1-4 and 31

The bacterial reverse mutation assay (Ames Test) was used to evaluatethe mutagenic properties of tested Compounds 1-4 and 31. The test usedamino acid-dependent strains of Salmonella typhimurium and Escherichiacoli to detect point mutations which involved substitution, addition ordeletion of one or a few DNA base pairs. Point mutations were introducedin the histidine (Salmonella typhimurium) or the tryptophan (Escherichiacoli) operon, making the tester strains incapable of producing theseamino acids. The test detected mutations that revert the mutationspresent in the bacteria, restoring the functional capability tosynthesize histidine or tryptophan. The revertant bacteria were detectedby the ability to grow in the absence of the amino acid required by theparent test strain. A mutagenic potential of a tested compound wasassessed by exposing these bacterial strains to different concentrationsof the compounds and estimating number of revertant colonies grown inthe absence (trace quantities) of the amino acid.

A. Materials and Equipment

Cell strains used in the assay included the Salmonella typhimuriumstrains TA 98 (Xenometrix PSS-0110), TA 100 (Xenometrix PSS-0111), TA1535 (Xenometrix PSS-0112), and TA 1537 (Xenometrix PSS-0113), andEscherichia coli strains wp2 [pKM101] (Xenometrix PSS-0116) and wp2 uvrA(Xenometrix PSS-0115).

Reagents and consumables included: DMSO (Sigma Cat# 34869), DMSO stocksolution of the tested compound(s) at 45 mM, magnesium sulfate MgSO₄·H₂O(Fluka Cat# 83266), citric acid monohydrate (Enamine, Ukraine),potassium phosphate dibasic K₂HPO₄ (Helicon Cat# Am-0348), sodiumammonium phosphate Na₂NH₂PO₄ (Sigma Cat# S9506), D-glucose monohydrate(Sigma Cat# 49158), Agar (Sigma Cat# A1296), L-histidine (Sigma Cat#H6034), biotin (Sigma Cat# B4639), L-tryptophan (Sigma Cat# T8941),nutrient broth #2 (Oxoid Cat# CV0067), ampicillin (Sigma Cat# A9393),2-nitrofluorene (Sigma Cat#N16754), 4-nitroquinoline N-oxide (SigmaCat#N8141), 9-aminoacridine (Enamine, Ukraine; T5111202), sodium azide(Helicon Cat# Am-0639), sodium chloride (Sigma Cat# S3014), magnesiumchloride MgCl₂·6H₂O (Sigma Cat# M2670), 35 mm Petri dish (Corning Cat#430588), and 1.5 mL Eppendorf tubes (Greiner bio-one Cat# 616201).

The media used was Vogel-Bonner E medium (7.5 mM MgSO₄, 10 mM citricacid monohydrate, 60 mM K₂HPO₄, 15 mM Na₂NH₂PO₄); GM medium (glucoseminimal agar medium) (Vogel-Bonner E medium supplemented with 0.5%glucose, 1.5% agar); GM liquid medium (glucose minimal medium)(Vogel-Bonner E medium supplemented with 0.5% glucose); and Top agar(0.6% agar, 0.6% NaCl, 0.05 mM histidine and 0.05 mM biotin (for S.typhimurium) or 0.05 mM tryptophan (for E. coli)).

Equipment included an Innova 4080 Incubator Shaker (New BrunswickScientific, USA), a BioMate 3 UV/Vis spectrophotometer (ThermoScientific, USA), a Termaks Incubator, B8054 (Termaks, Norway), thewater purification system NANOpure Diamond D11911 (Thermo ScientificBarnstead, USA), and pipettors 2-20 µL, 20-100 µL, and 100-1000 µL(Thermo Scientific, USA).

B. Methods

Experiments with a test compound and positive and negative controls wereconducted in duplicate on five tester strains (S. typhimurium: TA 98, TA100, TA 1535, TA 1537; and E. Coli: wp2[pKM101] + wp2 uvrA mixed 1:2).As the positive controls, compounds with known mutagenic activity wereused: 2-nitrofluorene (0.1 µg/plate) for TA 98, 4-nitroquinoline N-oxide(0.02 µg/plate) for TA 100, NaN₃ (0.15 µg/plate) for TA 1535,9-aminoacridine (7.5 µg/plate) for TA 1537, and 4-nitroquinoline N-oxide(0.02 µg/plate) for E. coli. DMSO was used as the negative control.

For each experiment, tester strain cultures were grown overnight inOxoid nutrient broth #2 at 37° C. with shaking at 235 rpm to a densityof 1-2×10⁹ colony forming units/mL (OD₅₄₀~2).

The tester strain (10 µL of night culture) was mixed with the test agent(10 µL of the DMSO stock) and GM liquid medium (30 µL). The obtainedmixture was incubated at room temperature for 5 min in a sterile 1.5 mLtube. Then, to the tube was added 200 µL of molten top agar stored at44° C. to prevent solidifying. After mixing with a pipette, the mixturewas poured onto the surface of a GM agar plate (2.0 mL of GM agar perplate). After solidifying the top agar, the plate was inverted andincubated at 37° C. for 48 hours. Then, results were expressed as numberof revertant colonies per plate.

C. Results

The final concentrations of the tested compounds were determined to be50 µM, 100 µM and 200 µM. Precipitate was observed in astrain-compound-GM medium mixture in the samples with finalconcentrations of 200 µM for Compounds 1 and 4.

Tested Compounds 1, 2, 4 and 31 had no mutagenic potential for thetester strains. The test Compound 3 at the concentration of 200 µMrevealed mutagenic activity for the tester strains TA 98, TA 1537, TA1535 and E. coli. Compound 3 had mutagenic activity at both 100 and 200µM for the strain TA 100.

The results are shown in Table 4 below.

TABLE 4 Results of the mutagenicity assay for the tested compounds CellStrain Cone. (µM) Mean ± SD Fold increase (over baseline*) Mean ± SDFold increase (over baseline*) Mean ± SD Fold increase (over baseline*)Mean ± SD Fold increase (over baseline*) Mean ± SD Fold increase (overbaseline*) Compound 31 Compound 1 Compound 2 Compound 3 Compound 4 TA 980 2.5±0.7 2.5±0.7 2.5±0.7 2.5±0.7 2.5±0.7 200 1±1.41 0.3 0±0 0 0.5±0.70.2 >1000 >312 0±0 0 100 1±0 0.3 0.5±0.7 0.2 0.5±0.7 0.2 0.5±0.7 0.2 0±00 50 0.5±0.7 0.2 1.5±0.7 0.46 1±1.41 0.3 1.5±0.7 0.46 1.5±0.7 0.46positive 7±1.41 7±1.41 7±1.41 7±1.41 7±1.41 TA 100 0 5.5±0.7 5.5±0.75.5±0.7 5.5±0.7 5.5±0.7 200 6±1.41 0.97 0±0 0 10±0 1.61 >300 >48 4±1.410.65 100 6±0 0.97 0±0 0 6±2.83 0.97 >200 >32 7.5±3.54 1.2 50 6.5±0.71.04 0±0 0 8.5±6.36 1.37 5.5±0.7 0.85 5.5±2.12 0.89 positive 189.5±19.09189.5±19.09 189.5±19.09 189.5±19.09 189.5±19.09 TA 1535 0 3±1.41 3±1.413±1.41 3±1.41 3±1.41 200 2.5±0.7 0.57 0±0 0 1±0 0.23 >150 >34 0±0 0 1001±0 0.23 0±0 0 3±0 0.68 0±0 0 1.5±0.7 0.34 50 2.5±2.12 0.57 1±0 0.23 0±00 1.5±0.7 0.34 1±0 0.23 positive 307.5±10.6 307.5±10.6 307.5±10.6307.5±10.6 307.5±10.6 TA 1537 0 1.5±0.7 1.5±0.7 1.5±0.7 1.5±0.7 1.5±0.7300 0.5±0.7 0.23 1±0 0.45 0.5±0.7 0.23 12.5±4.94 5.68 0.5±0.7 0.23 2000±0 0 1±1.41 0.45 1±0 0.45 0.5±0.7 0.23 0±0 0 100 1±1.41 0.45 0.5±0.70.23 1.5±2.12 0.68 1±1.41 0.45 0.5±0.7 0.23 50 1119±21.21 1119±21.211119±21.21 1119±21.21 1119±21.21 positive 4±0 4±0 4±0 4±0 4±0 0 0±0 00±0 0 2±2.82 0.5 >300 >75 5.5±2.12 1.38 200 1.5±0.7 0.38 0±0 0 1.5±0.70.38 7±0 1.75 1.5±0.7 0.38 E. coli 100 4.5±3.53 1.13 0±0 0 2.5±2.12 0.634±0 1 3±1.41 0.75 50 13±0 13±0 13±0 13±0 13±0 positive 2.5±0.7 2.5±0.72.5±0.7 2.5±0.7 2.5±0.7 *Baseline - mean of the negative control + SD

Example 3: Assessment of Cytotoxicity for Compounds 1-4 and 31

Compounds 1-4 and 31 were tested in a cytotoxicity assay. The assay wasbased on the conversion of the non-fluorescent dye resazurin to thefluorescent compound rezorufin in the reducing environment of livingcell cytoplasm. The more living cells in a sample result in higherfluorescence, while less living cells result in lower fluorescence.

A. Materials and equipment

Reagents and consumables included: DMSO Chromasolv Plus, HPLC grade,≥99.7% (Sigma-Aldrich, USA; Cat #34869), DMEM (4.5 g/L) liquid withoutL-Glutamine (PAA, UK; Cat# E15-009), Dulbecco’s PBS (1x) without Ca andMg (PAA, UK; Cat# H15-002), L-glutamine (200 mM) (PAA, UK; Cat#M11-004), Fetal Bovine Serum “GOLD” EU approved (PAA, UK; Cat# A15-151),penicillin/streptomycin (100x) (PAA, UK; Cat# P11-010), resazurin(SynbiaS, Ukraine; Cat# 62758-13-8), doxorubicin, valium for solutionfor injection (Arterium, Ukraine; pharmaceutical), trypsin EDTA (10x)0.5% / 0.2% in DPBS (PAA, UK; Cat# L11-003), Costar ® 96-well cellculture cluster round bottom with polystyrene lid (Corning Incorporated,Cat# 3790), disposable pipettor tips (Thermo Scientific, Fisherbrand,Eppendorf USA), centrifuge tubes, 50 mL (Santa Cruz, USA; Cat#sc-200251), Falcon ® 96-well plate, black/clear (BD, Cat# 358078) , andserological pipettes 5 mL, 10 mL, 25 mL (Greiner Bio-One).

Equipment included: a cell culture CO₂ incubator, model CCL-170B-8(ESCO, Singapore), a centrifuge 5804R (Eppendorf, USA), an etchedhemacytometer, dark-line counting chamber (Hausser Scientific, USA;Cat#3500), CyBi®-SELMA, semiautomatic 96-fold pipettor (Analytik JenaAG), a Labculture Biological Safety Cabinet, Class II, Type A2 (ESCO),an inverted Microscope, Model CK2 (Olympus Optical Co., Ltd., Japan), amicroscope Leica DM LS2 (Leica Microsystems Wetzlar GmbH, Germany), amulti-mode microplate reader POLARstar Omega (BMG Labtech GMBH,Germany), a Titertek Multidrop 384 Model 832 (Thermo Scientific /Titertek, USA), StakMax Microplate Handling System (Molecular Devices),PIPETMAN pipettes 2-20 µL, 50-200 µL, 200-1000 µL (Gilson, USA), andmultichannel electronic pipettes 2-125 µL, 5-250 µL, 15-1250 µL, Matrix(Thermo Scientific, USA).

B. Methods

HepG2 cells were cultivated in a humidified atmosphere at 37° C. and 5%CO₂ in 75 cm² flasks to 80 - 90% of confluence and split twice per weekwith a subcultivation ratio of 1:4. The cell layer was rinsed with PBSto remove all traces of serum. Then 3.0 mL of solution containing 0.25%(w/v) trypsin and 0.53 mM EDTA was added to the flask and incubatedabout 10 minutes. The HepG2 cells were detached using a scraper andre-suspended in DMEM containing 10% FBS and 2 mM glutamine. The cellsuspension with a final concentration of 5×10⁵ cells/mL was dispensedinto sterile 96-well black wall clear flat bottom plates as previouslydescribed (McMillian et al. (2002) Cell Biol. Toxicol. 18(3):157-173;Mulvihill et al. (2009) Future Med. Chem. 1(6):1153-1171) and the testcompounds were added.

The compound DMSO stock solutions were diluted with PBS to achieve anintermediate concentration of 1 mM. The cell proliferation assessmentwas performed at different concentrations of compounds ranging from 0.1µM to 100 µM. Doxorubicin at a final concentration of 20 µM was used asa positive control. After compound addition, cells were incubated for 24hours in a humidified atmosphere at 37° C. and 5% CO₂.

Resazurin was added to the final concentration (50 µM) and incubated for4 hours under the same conditions. Presence of rezorufin (cellviability) was quantified by measuring fluorescence (Ex - 490 nm, Em -540 nm; see Vega-Avila and Pugsley (2011) Proc. West Pharmacol. Soc.54:10-14).

The percent of cell proliferation inhibition was calculated by applyingthe following formula:

$\text{CPI}\,\text{=}\left( \frac{\text{AVG}_{High\, controls} \sim \,\text{Well}_{RFU}}{\text{AVG}_{High\, controls} \sim \,\text{AVG}_{Low\, controls}} \right)\, \ast \, 100$

where:

-   CPI - cell proliferation inhibition;-   AVG High control - mean of RFU of wells containing cell suspension    and PBS;-   Well RFU - RFU of target well with test compound; and-   AVG Low controls - mean of RFU of wells containing cell suspension    and doxorubicin.

IC₅₀ values were calculated using GraphPad Prism software.

C. Results

Doxorubicin was used as a reference compound to assess inhibition ofcell proliferation. The data of HepG2 cell proliferation inhibition bydoxorubicin at 20 µM were used as high control and the value of RFU wastaken for 100% of inhibition.

HepG2 cell proliferation inhibition (%) by the Compounds 1-4 and 31 islisted in Table 5 below.

TABLE 5 HepG2 cell proliferation inhibition (%) by the Compounds 1- 4and 31. Concentration of compounds, µM HepG2 proliferation inhibition(%) 21 1 2 3 4 100 µM -17.7 0.1 14.7 0.5 -5.5 30 µM -5.4 -2.5 2.4 -0.1-3.8 10 µM -2.2 -3.9 2.6 2.6 -1.4 3 µM -0.9 -1.8 5.6 3.9 5.4 1 µM -1.5-0.6 1.2 2.4 0.8 0.3 µM -0.8 -2.0 2.8 0.2 0.1 0.1 µM 2.0 -0.1 -1.8 -2.1-1.8

Based on the results of the study of proliferation inhibition of HepG2cells at different concentrations ranging from 100 µM to 0.1 µM, none ofthe tested compounds exhibited significant cytotoxic effects.

Example 4: In Vitro Predictor hERG Fluorescence Polarization Assay forCompounds 1-4 and 31

A preliminary assessment of human Ether-a-go-go-Related Gene (hERG)binding for Compounds 1-4 and 31 a fluorescence polarization assay wasperformed. Fluorescence polarization (FP) readout technology is based onthe observation that when a small fluorescent molecule (the tracer) isexcited by the polarized light, the emitted light is largely depolarizedbecause of the rapid rotation of the molecule in the solution during itsfluorescence lifetime. The hERG predictor assay provides valuableinformation about the possible binding of test compounds to thepotassium channel and potential QT prolongation on echocardiogram.

A. Materials and Equipment

Reagents and consumables used included DMSO Chromasolv Plus, HPLC grade,≥99.7% (Sigma-Aldrich, USA; Lot # 34869), Predictor™ hERG FluorescencePolarization Assay kit (Invitrogen; Cat#PV5365), Corning assay plate,384 wells, U-bottom, black polystyrene (Corning, USA; Cat.# 3677), andCompounds 1-4 and 31.

Equipment included a TECAN ULTRA Multifunctional Plate Reader (Tecan,Austria), micropipettes 0.5-5 µL, 2-20 µL,15-200 µL, 100-1000 µL(Finntip, Eppendorf, Gilson), a multichannel pipette (30 µL) (ThermoMatrix, USA), and a water purification system, NANOpure Diamond D11911(Thermo Scientific Barnstead, USA).

B. Methods

All experiments were performed using the Predictor™ hERG FluorescencePolarization Assay in accordance with the manufacturer’s protocol PV5365(Invitrogen, Carlsbad, CA).

The hERG reaction was performed by incubating the tracer and membraneswith hERG channel for 2-4 hours in the solution. The fluorescencepolarization was maximal when nothing interfered with the reaction ofthe tracer and hERG membranes (minimal tracer rotation). But when atested compound competed with the tracer for the hERG channel, thepolarization of emitted light lowered due to the ability of free unboundtracer to rotate rapidly in the solution. The reference compound(E-4031, provided by the manufacturer) was used to validate assayperformance. The calibration curve of the E-4031 was used to compare theIC₅₀ of E-4031 in the performed assay with the manufacturer’s provideddata. “Sigmoidal dose-response (variable slope)” function of GraphPadPrism software was used for the calibration curve building andcalculation of IC₅₀ for E-4031 assessment. The IC₅₀ value for E-4031 wasfound to be approximately 70 nM in accordance with the published data.

All test points for the compounds were performed in quadruplicates.Three dilutions of the tested compounds were assessed - 1 µM, 5 µM and20 µM.

A set of positive and negative controls (Assay blank - no tracer added,Assay Negative - 30 µM of E-4031 that represented 100% tracerdisplacement and gave minimum assay polarization value) was performedwith 4 repeats.

C. Results

Compounds 1, 2, 3 and 31 showed significant and dose dependentinhibition of the tracer binding, suggesting possible presence of hERGliability. Compound 4 showed low inhibition of the tracer bindingwithout dose dependence. See Table 6 below and FIG. 18 .

TABLE 6 hERG binding profile for Compounds 1-4 and 21 Binding values, %Mean SE Compound 31, 20 µM 92.6 80.0 87.8 90.2 88 2.7 Compound 31, 5 µM73.4 78.2 68.6 73.4 73 2.0 Compound 31, 1 µM 47.6 48.8 41.0 44.6 45 1.7Compound 1, 20 µM 105.3 102.3 99.2 105.3 103 1.4 Compound 1, 5 µM 99.298.0 100.5 99.2 99 0.5 Compound 1, 1 µM 81.2 87.2 79.4 83.6 83 1.7Compound 2, 20 µM 92.6 92.0 90.8 90.2 91 0.5 Compound 2, 5 µM 69.8 80.669.8 75.8 74 2.6 Compound 2, 1 µM 44.0 49.4 45.2 49.4 47 1.4 Compound 3,20 µM 98.6 98.6 102.3 97.4 99 1.0 Compound 3, 5 µM 111.9 117.3 113.7118.5 115 1.5 com Compound 42.2 49.4 26.6 45.8 41 5.0 Compound 4, 20 µM17.0 25.4 27.2 18.8 22 2.5 Compound 4, 5 µM 25.4 27.8 27.2 29.0 27 0.8Compound 4, 1 µM 22.4 32.0 24.2 27.2 26 2.1 E4031,30 µM(ref) -C 99.8101.1 98.6 100.5 100 0.5 +C -5.9 3.8 -5.3 7.4 0 3.3

Example 5: Assessment of Caco-2 A-B Permeability for Compounds 1-4 and31

A Caco-2 permeability assay was performed to determine the suitabilityof Compounds 1-4 and 31 for oral dosing by predicting the in vivoabsorption of drugs in the intestine by measuring the rate of transportof the compound across the Caco-2 cell line.

A. Materials and Equipment

Reagents and consumables used included: Trypsin EDTA (10x) 0.5% / 0.2%in DPBS (PAA, UK; Cat# L11-003), HEPES, High Purity Grade (Helicon,Am-0485), Dulbecco’s PBS (1x) without Ca and Mg (PAA, UK; Cat# H15-002),Hanks' BSS (1x) without Ca and Mg and without phenol red (PAA, UK; Cat#H15-009), DMSO Chromasolv Plus, HPLC grade, ≥99.7% (Sigma-Aldrich, USA;Cat #34869), DMEM (4.5 g/L) liquid without L-glutamine (PAA, UK; Cat#E15-009), L-glutamine (200 mM) (PAA, UK; Cat# M11-004), Fetal BovineSerum “GOLD” EU approved (PAA, UK; Cat# A15-151),penicillin/streptomycin (100x) (PAA, UK; Cat# P11-010), acetonitrileChromasolv, gradient grade, for HPLC, ≥99.9% (Sigma-Aldrich, USA; Cat#34851), formic acid for mass spectrometry, ~98% (Fluka, USA; Cat#94318), Falcon® HTS 24-multiwell insert systems with media feeder tray(BD Biosciences, USA; Prod# 351181), Falcon® 24-well TC-treated cell PSpermeable support companion plate (BD, Prod# 353504), centrifuge tubes,50 mL (Santa Cruz, USA; Cat# sc-200251), serological pipettes 5 mL, 10mL, 25 mL (Greiner Bio-One), disposable pipettor tips (ThermoScientific, Fisherbrand, Eppendorf USA), 1.1 mL microtubes in microracks(Thermo Scientific, USA), Zorbax Eclipse Plus C18 column 2.1×50 mm, 3.5µm (Agilent Technologies, Inc. USA), propranolol hydrochloride ≥99%(TLC), powder (Sigma-Aldrich, USA; Cat # P0884), imipraminehydrochloride ≥99% (TLC) (Sigma-Aldrich, USA; Lot # 17379), andatenolol, analytical reference material, ≥98.5% (HPLC) (Sigma-Aldrich,USA; Cat #74827).

Equipment included: a cell culture CO₂ incubator, model CCL-170B-8(ESCO, Singapore), a centrifuge 5804R (Eppendorf, USA), a centrifuge4-15C (Qiagen) (Sigma, Germany), an etched hemacytometer, dark-linecounting chamber (Hausser Scientific, USA; Cat#3500), a gradient HPLCsystem VP (Shimadzu, Japan), an Innova 4080 Incubator Shaker (NewBrunswick Scientific, USA), a Millicell-ERS system ohm meter (Millipore,Cat # MERS 000 01), an MS/MS detector API 3000 PE with TurboIonSprayElectrospray module (PE Sciex, USA), a multichannel manual pipette(Thermo Labsystems Finnpipette, FA16-50R), multichannel electronicpipettes 2-125 µL, 5-250 µL, 15-1250 µL, Matrix (Thermo Scientific,USA), PIPETMAN pipettes 2-20 µL, 50-200 µL, 200-1000 µL (Gilson, USA),VWR membrane nitrogen generators N2-04-L1466, nitrogen purity 99%+ (VWR,USA), and a water purification system NANOpure Diamond D11911 (ThermoScientific Barnstead, USA).

All measurements were performed using a Shimadzu VP HPLC system thatincluded a vacuum degasser, gradient pumps, reverse phase HPLC column,column oven and autosampler. The HPLC system was coupled with a tandemmass spectrometer API 3000 (PE Sciex). The TurboIonSpray ion source wasused in both positive and negative ion modes. Acquisition and analysisof the data were performed using Analyst 1.5.2 software (PE Sciex).

The LC-MS conditions were as follows. Column: Agilent ZORBAX EclipsePlus C18; Mobile phase A: Acetonitrile:Water:Formic acid = 100:1000:1;Mobile phase B: Acetonitrile:Formic acid = 1000:1; Gradient: 0 min 25%B, 1.1 min 100% B, 1.5 min 100% B, 1.51 min 25% B, 2.7 min stop; Elutionrate: 400 µL/min; Column temperature: 30° C.; Injection volume: 2 µL;Ion source: Turbo spray; Ionization model: ESI; Scan type: Positive Q3Multiple Ions; Nebulize gas: 15 L/min, Curtain gas: 8 L/min; Ionsprayvoltage: 5000 V, Temperature: 400° C.

B. Methods

Caco-2 cells were cultivated in 75 cm² flasks to 80-90% of confluenceaccording to the ATCC and Millipore recommendations (Millipore protocolnote PC1060EN00P) in a humidified atmosphere at 37° C. and 5% CO₂. Cellswere detached with Trypsin/EDTA solution and re-suspended in the cellculture medium to a final concentration of 2×10⁵ cells/mL. 500 µL of thecell suspension was added to each well of an HTS 24-multiwell insertsystem and 35 mL of prewarmed complete medium was added to the feedertray. Caco-2 cells were incubated in the multiwell insert system for 10days before the transport experiments. The medium in the filter plateand feeder tray was changed every other day. After 10 days of cellgrowth, the integrity of the monolayer was verified by measuring thetransepithelial electrical resistance (TEER) for every well using theMillicell-ERS system ohm meter. The TEER values obtained were greaterthan 1000 Ω (between 1400 and 1500 Ω) as required by the assayconditions. The 24-well insert plate was removed from its feeder plateand placed in a new sterile 24-well transport analysis plate. The mediumwas aspirated and inserts washed with PBS.

To determine the rate of drug transport in apical (A) to basolateral (B)direction, 300 µL of the test compound solution in buffer (HBSS, 5.6 mMglucose, 10 mM HEPES, pH=7.4) was added into the filter wells and 1000µL of the same buffer was added to wells in the transport analysisplate. The plates were incubated for 90 min. at 37° C. with shaking at50 rpm. 75 µL aliquots were taken from the apical and basolateralcompartment for LC-MS/MS analysis. All samples for LC-MS/MS analysiswere extracted by acetonitrile (x2 volume) followed by proteinsedimentation by centrifuging at 10000 rpm for 10 minutes. Supernatantswere analyzed using the HPLC system coupled with a tandem massspectrometer.

Imipramine, propranolol (high permeability), and atenolol (lowpermeability) were used as reference compounds.

The apparent permeability (Papp) was calculated for the Caco-2permeability assay using the following equation:

P_(app) = (V_(A)/((Area)x(Time))x(|drug|_(acc)/|drug|_(initial, d), where:)

-   VA - volume of transport buffer in acceptor well;-   Area - surface area of the insert (equals to effective growth area    of the insert -0.31 sq.cm);-   Time - time of the assay;-   [drug]_(acc) - concentration of test compound in acceptor well;-   [drug/_(initial,d) - initial concentration of test compound in a    donor well; and-   Papp is expressed in 10⁻⁶ cm/sec.

C. Results

The A-B permeability data for Compounds 1-4 and 31 reference compoundsimipramine, propranolol, and atenolol is listed in Table 7 below. TheA-B permeability values for the reference compounds correspond to theliterature data (see, e.g., Lau et al. (2004) Drug Metab. Dispos.32:937-942; Fujikawa et al. (2005) Bioorg. Med. Chem. 13(15):4721-4732;Rubas et al. (1996) J. Pharm. Sci. 85(2): 165-169). The permeability ofall test compounds can be classified as medium to high permeability.

TABLE 7 A-B permeability data of Compounds 1-4 and 31 Compound IDPermeability (10⁻⁶ cm/s) 1 2 Mean SD Imipramine 21.1 17.8 19.5 2.4Propranolol 23.0 20.6 21.8 1.7 Atenolol 0.5 0.4 0.5 0.1 31 52.8 45.249.0 5.4 1 8.3 12.1 10.2 2.7 2 17.9 18.5 18.2 0.4 3 39.5 41.7 40.6 1.6 415.9 14.6 15.3 0.9

Example 6: Assessment of Metabolic Stability in Mouse Liver Microsomesfor Compounds 1-4 and 31

The metabolic stability of Compounds 1-4 and 31 and two referencecompounds (imipramine and propranolol) in liver microsomes wasdetermined at five time points over 40 minutes using HPLC-MS. Metabolicstability was defined as the percentage of parent compound lost overtime in the presence of a metabolically active test system, such asrodent liver microsomal fractions.

A. Materials and Equipment

Reagents and consumable used included: DMSO Chromasolv Plus, HPLC grade,≥99.7% (Sigma-Aldrich, USA; Cat #34869), acetonitrile Chromasolv,gradient grade, for HPLC, ≥99.9% (Sigma-Aldrich, USA; Cat #34851),potassium phosphate monobasic ACS Grade (Helicon, Cat # Am-O781),potassium phosphate dibasic ACS Grade (Helicon, Cat # Am-O705),magnesium chloride hexahydrate (Helicon, Cat # Am-O288), microsomes fromliver, pooled, male BALB/c mice, glucose-6-phosphate dehydrogenase frombaker’s yeast (S. cerevisiae), type XV (Sigma-Aldrich, USA; Cat #G6378), glucose-6-phosphate sodium salt, Sigma Grade, crystalline(Sigma-Aldrich, USA; Cat # G7879), NADPH tetrasodium salt, ≥95% (SantaCruz Biotechnology, Inc., USA; Cat # sc-202725), formic acid for massspectrometry, ~98% (Fluka, USA; Cat #94318), DMSO stock solutions of thetest compounds at 10 mM, propranolol hydrochloride ≥99% (TLC), powder(Sigma-Aldrich, USA; Cat # P0884), imipramine hydrochloride ≥99% (TLC)(Sigma-Aldrich, USA; Lot # I7379), Zorbax Eclipse Plus C18 column 2.1×50mm, 3.5 µm (Agilent Technologies, Inc. USA), and 1.1 mL microtubes inmicroracks, pipettor tips (Thermo Scientific, USA).

Equipment included: a gradient HPLC system VP (Shimadzu, Japan), anMS/MS detector API 3000 PE with TurboIonSpray Electrospray module (PESciex, USA), VWR membrane nitrogen generators N2-04-L1466, nitrogenpurity 99%+ (VWR, USA), Innova 4080 Incubator Shaker (New BrunswickScientific, USA), a water purification system NANOpure Diamond D11911(Thermo Scientific Barnstead, USA), a centrifuge 4-15C (Qiagen) (Sigma,Germany), and multichannel electronic pipettes 2-125 µL, 5-250 µL,15-1250 µL, Matrix (Thermo Scientific, USA; Cat ## 2001, 2002, 2004).

All measurements were performed using a Shimadzu VP HPLC systemincluding vacuum degasser, gradient pumps, reverse phase HPLC column,column oven and autosampler. The HPLC system was coupled with a tandemmass spectrometer API 3000 (PE Sciex). The TurboIonSpray ion source wasused in both positive and negative ion modes. Acquisition and analysisof the data were performed using Analyst 1.5.2 software (PE Sciex).

B. Methods

Mouse hepatic microsomes were isolated from pooled (50), perfused liversof BALB/c male mice according to the standard protocol (Hill, J.R. inCurrent Protocols in Pharmacology 7.8.1-7.8.11, Wiley Interscience,2003). The batch of microsomes was tested for quality control usingimipramine, propranolol and verapamil as reference compounds. Microsomalincubations were carried out in 96-well plates in 5 aliquots of 40 µLeach (one for each time point). Liver microsomal incubation mediumcontained PBS (100 mM, pH 7.4), MgCl₂ (3.3 mM), NADPH (3 mM),glucose-6-phosphate (5.3 mM), glucose-6-phosphate dehydrogenase (0.67units/mL) with 0.42 mg of liver microsomal protein per mL. Controlincubations were performed replacing the NADPH-cofactor system with PBS.Compounds 1-4 and 31 (2 µM, final solvent concentration 1.6%) were eachincubated with microsomes at 37° C. while shaking at 100 rpm.Incubations were performed in duplicates. Five time points over 40minutes were analyzed. The reactions were stopped by adding 12 volumesof 90% acetonitrile-water to incubation aliquots, followed by proteinsedimentation by centrifuging at 5500 rpm for 3 minutes. Supernatantswere analyzed using the HPLC system coupled with tandem massspectrometer. The elimination constant (k_(e1)), half-life (t_(½)) andintrinsic clearance (Cl_(int)) were determined in a plot of ln(AUC)versus time, using linear regression analysis:

k_(al) = −slope

$t_{y_{2}} = \frac{0.693}{k}$

$Cl_{int} = \frac{0.693}{t_{1/2}} \times \frac{\mu l_{incubation}}{mg_{microsomes}}$

In order to indicate the quality of the linear regression analysis, theR (correlation coefficient) values were provided. In some cases, thelast time point was excluded from the calculations to ensure acceptablelogarithmic linearity of decay.

C. Results

Mouse microsomal stability data for two reference compounds (imipramineand propranolol) and Compounds 1-4 and 31 are shown in FIGS. 19A-19D.Compounds 4 and 31 showed low stability, Compounds 1 and 2 exhibitedmoderate stability and Compound 3 showed high metabolic stability in themouse hepatic microsomal test system. “No cofactor” control dataindicated that the observed instability was primarily determined byCYP450 activity.

Example 7: In Vitro CYP450 Inhibition for Compounds 1-4 and 31

A preliminary assessment of inhibition of the major CYP450 panel (1A2,2C9, 2C19, 2D6, 3A4) by Compounds 1-4 and 31 at a single compoundconcentration (10 µM) was made. CYP450 inhibition profiling providesvaluable information regarding any possible drug-drug interactions for atest compound.

A. Materials and Equipment

Reagents and consumable used included: DMSO Chromasolv Plus, HPLC grade,≥99.7% (Sigma-Aldrich, USA; Cat #34869); acetonitrile Chromasolv,gradient grade, for HPLC, ≥99.9% (Sigma-Aldrich, USA; Cat #34851);P450-Glo™ Screening Systems (Promega Corp.) that included CYP 1A2 (Cat.# V9770), CYP 2C9 (Cat. # V9790), CYP 2C19 (Cat. # V9880), CYP 2D6 (Cat.# V9890), CYP 3A4 (Cat. # V9800), and Luciferin-PPXE (Cat. # V9910);Corning assay plate 384 wells (Corning, USA; Cat.# 3673); and Matrix96-well assay plates (Matrix, Thermo Scientific, USA; Cat.# 4919).

Equipment included: a multi-mode microplate reader POLARstar Omega (BMGLabtech GMBH, Germany); a dry thermostat CT50 (Ukrorgsynthez, Ukraine;CT 50); micropipettes 0.5-5 µL, 2-20 µL, 15-200, 100-1000 µL (Finntip,Eppendorf, Gilson); and multichannel electronic pipette 1.0-30 µL,Matrix (Thermo Scientific, USA; Cat # 2060).

B. Methods

All experiments were performed using P450-Glo^(Tm) Assay Systems(Promega) in accordance with the manufacturer’s protocols. TheP450-Glo^(Tm) Assays provide a luminescent readout-based method formeasuring cytochrome P450 activity. A conventional cytochrome P450reaction was performed by incubating the cytochrome P450 and aluminogenic cytochrome P450 substrate. The substrates in the P450-Glo™assays are derivatives of beetle luciferin. The derivatives themselvesare not substrates for luciferase but are converted by cytochrome P450sto luciferin, which in turn reacts with luciferase to produce light. Theamount of light produced was directly proportional to cytochrome P450activity. All test points were performed in quadruplicates. Controlmembranes (without CYPs) represented the Negative control (baseline).DMSO final concentration was 0.25%.

The following reference compounds were used to assess CYP inhibition:

CYP Reference inhibitor Ref. inhibitor conc., uM CYP inhibition, % 1A2alfa-naphthoflavone 4 99.64 2C9 fluconazole 120 84.46 2C19 omeprazole 2484.06 2D6 quinidine 1 88.42 3A4 ketoconazole 20 102.76

Concentrations of alfa-naphthoflavone, quinidine, ketoconazole,fluconazole and omeprazole are shown as 4x of Promega protocolrecommendations or 4x of their IC₅₀ found in the literature (see, e.g.,Li et al. (2004) Drug Metab. Dispos. 32(8): 821-827; Niwa et al. (2005)Biol. Pharm. Bull. 28(9): 1805-1808).

C. Results

The CYP inhibition profiles for Compounds 1-4 and 31 are shown below andin FIGS. 20A-20E. At the concentration of 10 µM, Compound 3 showed veryhigh inhibition of all 5 tested CYP450. CYP 1A2 was significantlyinhibited by Compounds 1 and 2. CYP 2D6 was significantly inhibited byCompound 31. Compound 4 did not show a significant inhibition of anyCYP450 isoform.

1 CYP inhibition profile for Compound 31 CYP Inhibition, % Mean SE 1A216.7 -0.7 3.6 -22.5 -0.7 8.1 2C9 50.4 -18.3 11.9 13.7 14.5 14.1 2C1914.0 18.2 14.4 18.2 16.2 1.2 2D6 41.0 75.7 66.5 59.0 60.6 7.4 3A4 -17.94.1 -34.5 -16.1 11.2

2 CYP inhibition profile for Compound 1 CYP Inhibition, % Mean SE 1A273.2 60.1 69.6 73.2 69.0 3.1 2C9 40.6 11.1 15.7 28.5 24.0 6.6 2C19 5.07.4 11.6 13.5 9.4 1.9 2D6 18.5 13.7 18.8 17.0 1.6 3A4 -6.9 -37.2 -29.0-24.4 9.1

3 CYP inhibition profile for Compound 2 CYP Inhibition, % Mean SE 1A249.3 30.4 47.1 34.8 40.4 4.6 2C9 36.6 30.3 25.4 21.4 28.4 3.3 2C19 -25.0-8.6 0.8 -5.3 -9.5 5.5 2D6 25.3 0.5 26.0 17.3 8.4 3A4 -53.8 12.4 -1.4-14.3 20.2

4 CYP inhibition profile for Compound 3 CYP Inhibition, % Mean SE 1A298.6 96.4 95.7 100.0 97.6 1.0 2C9 96.2 98.8 98.0 97.4 97.6 0.5 2C19 97.489.9 94.6 88.0 92.5 2.1 2D6 98.9 100.3 99.6 99.9 99.7 0.3 3A4 64.8 86.9103.4 97.9 88.3 8.5

5 CYP inhibition profile for Compound 4 CYP Inhibition, % Mean SE 1A212.3 9.4 15.9 -15.2 5.6 7.1 2C9 27.3 11.9 4.6 -11.1 8.2 8.0 2C19 -11.4-11.8 -4.3 -13.7 -10.3 2.1 2D6 24.3 20.2 10.7 18.4 4.0 3A4 -4.1 37.2-29.0 1.4 19.3

Experimental Methods for Examples 8-11 Below Are as Follows Cell cultureand treatments

All cells were maintained at 37° C. under humidified conditions. CCCP(Sigma-Aldrich) was added to the culture media as a 2x stockconcentration. Test compounds 1, 2, 3, 4, and 31 were synthesized byEnamine and dissolved in DMSO (Sigma-Aldrich) at a stock concentrationof 10 mM. Aliquots were stored at -80° C. HeLa cells (ATCC) and HeLacells stably expressing untagged Parkin, EGFP-Parkin, 3xFLAG-ParkinC431S or EGFP-Parkin and mitoKeima were cultured in DMEM (Invitrogen)containing 10% FBS (BioWest). Rat adrenal pheochromocytoma cells (PC12)cells were grown in RPMI with 5% FBS and 10% horse serum. Fordifferentiation, cells were washed in PBS and plated on collagen coatedplates in low serum media containing 1% horse serum. Cells weredifferentiated for 14 days by addition of 100 ng/mL nerve growth factor(NGF).

Primary fibroblasts (Cell Applications) were cultured in fibroblastgrowth medium (FGM): DMEM containing 10% FBS, 1% penicillin-streptomycinand 1% non-essential amino acids. Direct conversion to neurons wasperformed utilizing short hairpin RNA targetingpolypyrimidine-tract-binding protein (shPTB), which affects proneuronalmicro-RNA circuits (Xue et al., Cell (2013) 152:82-96. Cells were seededwith 40,000 cells/mL and allowed to adhere overnight. 48 h aftertransduction with pLK0.1_shPTB lentivirus, positive cells were selectedwith 2 µg/mL puromycin in fibroblast growth medium. On day 6 media waschanged to FGM containing 10 ng/mL fibroblast growth factor (FGF,Genscript). On day 8, media was changed to differentiation media(DMEM:F12 (Invitrogen), 25 µg/mL insulin, 50 µg/mL transferrin, 0.1 µMputrescine 0.03 µM Na-selenite (all Sigma-Aldrich), and 15 ng/mL FGF)containing 5% FBS. On days 10 and 12 media was replaced withdifferentiation media containing reduced serum (2% FBS). On day 14,media was changed to differentiation media containing 2% FBS and 0.01µg/mL BDNF, GDNF, CNTF and NT3 (all peprotech) and 2% of anti-oxidantfree B27 (Invitrogen) for 48 h. Experiments were performed on day 16 indifferentiation media containing growth factors and B27.

Sandwich ELISA Assays

For ELISA assays, 96-well standard bind plates (Mesoscale Discovery)were coated with antibodies (FLAG, Sigma, F3165 or pSer65-Ub, both1:250) in bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.4)overnight and blocked with 5% BSA in TBS containing 0.02% Tween-20(TBST). For Parkin Ub-charging, cell lysates were prepared in RIPAbuffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate,0.1% sodium dodecyl sulphate (SDS)). 25 µg of lysates were added to theplates and incubated for 2 h. To quantify pSer65-Ub, cell lysates wereprepared in NP-40 buffer (50 mM Tris pH 7.6, 150 mM NaCl, and 0.5%NP-40) and diluted to 0.18% NP-40 with 1% BSA in TBST. 6.25 µg oflysates were added to the plates overnight and incubated at 4° C. Afterprimary antibody incubation, plates were washed and incubated with mousedetection antibodies (Ub, CST, #3933 or Ub, Millipore, MAB150 both1:250) for 1 h at RT. Secondary sulfo-tag conjugated antibodies wereincubated for 1 h in 1% BSA/TBST before plates were measured in 2x READbuffer using a Mesoscale Discovery Sector Imager 2400 (both MesoscaleDiscovery).

Immunofluorescence

Cells were grown on poly-D-lysine coated glass coverslips. Aftertreatments, cells were fixed in 1% paraformaldehyde and permeabilizedusing 1% Triton X-100, for each 10 min. Cells were blocked in 10% goatserum. Primary and secondary antibodies were diluted in 1% BSA/PBS andeach incubated for 1 h at RT. Primary antibodies used: DNA (mouse,1:250, Progen, AC-30-10), NBR1 (mouse, 1:100, AbnovaH00004077-M01),NDP52 (rabbit, 1:400, Proteintech, 12229-1-AP), OPTN (mouse, 1:200,Santa Cruz, sc-166576), p62 (mouse, 1:400, BD Biosciences, 610832),pSer65-Ub (rabbit, 1:500, in-house (Fiesel et al., EMBO Reports (2015)16:1114-30; Fiesel et al., (2015) Autophagy 11:2125-2126)), TAX1BP1(rabbit, 1:200, Cell Signaling, #5105), TOM20 (mouse, 1:100, Santa Cruz,sc-17764), TOM20 (rabbit, 1:2000, Proteintech, 11802-1-AP). Secondaryantibodies conjugated to AlexaFluor-568 or -647 (Invitrogen) werediluted 1:1000. Hoechst 33342 (Invitrogen) was diluted 1:5000 tocounterstain the nuclei.

Western Blot

Cells were harvested in RIPA buffer or NP-40 lysis buffer containingprotease and phosphatase inhibitors (Complete and PhosStop, RocheApplied Science) with the exception of Parkin Ub charging experimentswhere preheated (95° C.) SDS lysis buffer (50 mM Tris pH 7.6, 150 mMNaCl, 1% SDS) was used. Concentration of cell lysates was determinedwith bicinchoninic acid (Pierce Biotechnology). For Parkin Ub chargingexperiments, aliquots of lysates were treated with 0.1 M NaOH for 1 h at37° C. pH was neutralized with equinormal HCl.

Protein was subjected to SDS-PAGE using 4-20% or 8-16% Tris-Glycine gels(Invitrogen) and transferred onto polyvinylidene fluoride (PVDF)(Millipore). Membranes were incubated with primary antibodies overnightat 4° C. followed by HRP-conjugated secondary antibodies (1:10,000;Jackson ImmunoResearch Laboratories). Primary antibodies used: beta IIItubulin (rabbit, 1:1000, CST, #5568, CST), FLAG (mouse, 1:250,000,Sigma, F3165), GAPDH (mouse, 1:100,000-500,000, Meridian Life science,H86504M), GST (1:10,000, Sigma, G7781), MFN1 (mouse, 1:5,000, Abcam,ab57602), MFN2 (mouse, 1:5,000, Abcam, ab56889), Parkin (mouse, 1:2,000,Cell Signaling, #4211), pSer65-Ub (rabbit, 1:15,000, in-house (Fiesel etal., EMBO Reports (2015) 16:1114-30; Fiesel et al., (2015) Autophagy11:2125-2126)), TOM70 (rabbit, 1:5,000, Proteintech, 14528-1-AP), UBE2L3(rabbit, 1:10,000, ProteinTech, 14415-1-AP), VDAC1 (mouse, 1:5,000,Abcam, ab14734), vinculin (mouse, 1:100,000, Sigma, V9131).

Mitochondrial Depolarization Assay

Mitochondrial depolarization was assessed using a JC10 assay kit (Sigma,MAK159). HeLa Parkin cells were seeded with 70,000 cells/well in 20 µLvolume into 384-well plates with clear bottom. The following day 5 µL ofCCCP or compounds were added as 5x solutions. Equal volume of DMSOserved as a negative control. Cells were incubated for 4 h before 12.5µL of JC10 dye diluted in buffer A was added to the wells. One hourlater the reaction was stopped with 12.5 µL of buffer B and the platewas measured immediately on a Spectramax M5 plate reader (Moleculardevices) with bottom read using dual fluorescence (green: Ex 485 nm, Em538 nm, cut-off at 515 nm; red: Ex 544 nm, Em 590 nm, cut-off at 570nm). Mitochondrial depolarization was assessed by building the green tored ratio.

Example 8: Enzymatic Activation of Parkin in HeLa Cells

Functional testing of compounds was performed using an establishedprimary HCI assay for Parkin translocation, which correlates well withenzymatic activation (Fiesel et al., J. Cell Science (2014)127:3488-3504; Fiesel et al., Human Mutation (2015) 36:774-786). Inbrief, 1400 HeLa cells stably expressing EGFP-Parkin were seeded intooptical 384-well plates (Greiner BioOne) in 25 µL media and allowed toadhere for 40 h. Compounds 1, 2, 3, 4, and 31 were added to the plate as2x concentrated stocks. Control wells were treated with equal volume ofDMSO in media. After 2 h, unless otherwise stated, CCCP was added as 2xconcentrated stock with a final assay concentration of 10 µM for PCwells and 3.5 µM for test and NC wells. This low dose of CCCP wasexperimentally determined by a dose response curve of CCCP to result inno Parkin translocation (FIG. 13 ). After another 2 h, cells were fixedin 4% paraformaldehyde for 10 min and stained with Hoechst 33342(1:5000, Invitrogen) before plates were imaged and analyzed.

Image acquisition was performed on a BD Pathway 855 with the AttovisionV1.6 software (BD Biosciences). Wells were imaged with a 20x objectiveusing a 2×2 montage with laser autofocus. EGFP exposure time was 0.0175sec, Hoechst 0.0015 sec. Raw images were not processed and directlyanalyzed using a build-in ‘RING - 2 output’ algorithm. Values wereexported and normalized using JMP 11 software and transferred toGraphPad Prism 7 for quality control, graphing and curve fitting forDRCs with a variable slope using four parameters. The primary screeningassay typically showed S/B of 3.0-3.5 with CV of less than 5%. Valueswere normalized to both PC and NC values from 24 wells each per plateand the Z'score was calculated. Plates with Z' < 0.5 were repeated.

High-resolution imaging confirmed enhanced Parkin co-localization withmitochondria after treatment with compounds 1, 2, 3, 4, and 31 and a lowdose CCCP. This combination also induced the pSer65-Ub mitophagy tag asdetected by antibody staining (Fiesel et al., EMBO Reports (2015)16:1114-30; Fiesel et al., Autophagy (2015) 11:2125-2126) and furtherquantified by HCI in DRC format. Compared with Parkin translocation,EC₅₀ values for pSer65-Ub signal amplification were similar. Yet,compound wells with higher compound concentrations showed exceedingpSer65-Ub levels compared to PC wells (FIG. 10 ). No noticeable effecton cell survival was observed for any of the compounds even at highconcentrations. Neither compound alone nor low dose CCCP treatment (NC)was sufficient to activate Parkin translocation or induce pSer65-Ubsignal amplification, even after prolonged time points.

As readout for its activation, a catalytic site mutant that can be usedto trap Ub-charged Parkin was employed. Using HeLa cells stablyexpressing 3xFLAG-Parkin C431S, and as indicated by an upwards shiftedband, Ub-charged Parkin was generally increased with either compound asseen by western blot or by sandwich ELISA. To further validate effectson enhanced activation, we used HeLa cells expressing untagged nativeParkin. PC treatment with 10 µM CCCP induced pSer65-Ub signals and thecomplete degradation of some substrates (e.g., Mitofusins) while themost abundant, VDAC1, was still ubiquitylated (FIG. 8 ). Similar effectswere seen when cells had been pre-incubated with 5 µM of compound beforea low dose CCCP, which had no effect on its own.

Example 9: Downstream Mitophagy Activation in HeLa Cell Lines

To validate the activation of Parkin further downstream in the pathway,co-recruitment of endogenous autophagy receptors to mitochondria in HeLaEGFP-Parkin cells was analyzed. These dual adaptors recognize pSer65-Ubchains on damaged mitochondria and facilitate their engulfment byautophagosomal membrane via their interaction with LC3 proteins (Lazarouet al., Nature (2015) 524:309-14; Heo et al., Mol. Cell (2015) 60:7-20).

For pSer65-Ub analysis (Puschmann et al., Brain (2017) 140:98-117(2017); Fiesel et al., EMBO Reports (2015) 16:1114-30 (2015); Ando etal., Mol. Neurodegen. (2017) 12:32), EGFP-Parkin HeLa cells were seededand treated as above. After fixation, cells were permeabilized with 1%Triton X-100 in PBS for 10 min and blocked in 10% goat serum for 1 h. 20µL of primary anti-pSer65-Ub antibody (1:500 in 1% BSA in PBS) wereadded per well and incubated for 1 h. After washing, goat anti-rabbitAlexaFluor-568 antibody was added for 1 h. Nuclei were counterstainedwith Hoechst 33342 (1:5000 in PBS) before plates were imaged asdescribed above with additional acquisition for red fluorescence(exposure time 0.05 sec). pSer65-Ub signal was analyzed aftersegmentation as above as the signal intensity in the cytoplasmic ring.

All five compounds (1, 2, 3, 4, and 31) increased co-localization ofOPTN and of NBR1, NDP52, p62, and TAX1BP1 with Parkin on damagedmitochondria. Concomitant with enhanced recruitment of autophagyadaptors, a robust reduction of mitochondrial DNA in immunofluorescencestaining was observed.

To confirm mitophagy, HeLa cells expressing the pH-sensitive,mitochondria-targeted reporter mitoKeima46 were employed. Acharacteristic shift in the excitation wavelength of mitoKeima uponlysosomal fusion was used to determine mitochondrial turnover using HCI.For mitoKeima experiments (Kim et al., Mol. Neurodegen. (2016) 11:55),cells were seeded in DMEM media lacking phenol red in 20 µL per well.Hoechst counterstain was added as a 5x stock in 5 µL volume. Cells weretreated with 2x stock solutions of compounds and of CCCP as above. Equalamounts of DMSO were used for controls. Cells were imaged live andautofocus was used for each time point. Exposure times of neutral andacidic mitoKeima were 0.02 and 0.05 sec, respectively. Segmentation ofcells was performed as above and for each region of interest the ratioof acidic and neutral mitoKeima was calculated. Values were normalizedto the positive and negative controls.

Treatment with compound 4 in combination with various CCCPconcentrations (≥ 1 µM) resulted in a dose and time-dependent increaseof mitophagy, similar to the high dose positive control (FIG. 9 ). Peakvalues were obtained after 8 h of treatment indicating that a portion ofthe mitochondria had already been removed. Cells treated with low doseCCCP alone (up to 3.5 µM), did not show any increase of mitoKeimaacidification even at later time points. To determine EC₅₀ values forthe mitophagy assay, compounds 1, 2, 3, 4, and 31 were tested in DRCformat. Curve fit of data points after 4 and 8 h resulted in the sametrend in terms of potency within the group as the other quantitativeassays (FIG. 10 ). The activities of the compounds were in the nano- tomicromolar range, comparable with their potency in the Parkintranslocation and pSer65-Ub amplification assays

Example 10: Parkin Activation in Primary Cells and Neuronal Cultures

To validate Parkin activating drugs in primary cells, compound 4 wastested in combination with different low dose CCCP concentrations inhuman dermal fibroblasts. Similar to HeLa cells, 3.5 µM CCCP alone wasnot sufficient to induce a mitophagy response. When cells werepretreated with compound 4 followed by a low dose CCCP, enhancedubiquitylation of the Parkin substrates MFN1/2 was observed (FIG. 15A).Consistently, treatment with compound 4 significantly induced pSer65-Ublevels in the presence of low dose CCCP (FIG. 18A). This NCconcentration was used to test all five compounds side by side and foundthat while all five compounds induced a response to a certain extent,compounds 1 and 4 showed the strongest effects on MFN1/2 degradation andpSer65-Ub induction (FIG. 15B). In the absence of CCCP, the compoundsdid not induce any mitoQC response in primary fibroblast. Additionally,compounds with concentrations ten times higher than the EC₅₀ for Parkintranslocation did not induce a mitoQC response (FIG. 16B).

Next, induced Neurons (iNeurons) were generated from the fibroblastcultures. Neuronal conversion was confirmed by the neuronal markerbeta-III-tubulin. While there was only a subtle effect on MFN1ubiquitylation, all five compounds robustly induced pSer65-Ub levels(FIG. 15C). To ensure that compounds could also be used in an animalmodel, their ability to activate endogenous Parkin in rat PC-12 cellswas tested. All five compounds robustly induced pSer65-Ub inundifferentiated (FIG. 15D and FIG. 16C) and neuronal differentiatedcells (FIG. 15D), although here 3.5 µM CCCP alone did induce pSer65-Ubslightly. Nevertheless, cells pretreated with compounds showed morepronounced substrate ubiquitylation and degradation comparable to PCcells that were treated with 10 µM CCCP (see FIG. 15C and FIGS.16C-16D).

Example 11: In Vitro Enzyme Activity and Drug Binding

To demonstrate on-target effects of the compounds, in vitro assays withrecombinant, purified Parkin protein were performed. As the firstreadout for Parkin activity, E2 Ub discharge assays were performed,complementary to the Ub-charging assays of Parkin C431S in cells. UbcH7was first loaded with Ub and then mixed with Parkin that had beenpre-incubated with compound 4 or DMSO as control. Only in the presenceof PINK1, compound 4 led to more E2 discharge compared to control,consistent with enhanced Ub transfer onto the E3 enzyme (FIG. 15E). Thein vitro E2 discharge assays were performed as follows.

Ub-charging of E2 enzyme was performed for 90 min at 30° C. in areaction containing 0.1 µM GST-E1, 3.3 µM E2/UbcH7, 10 µM FLAG-Ub and0.188 µM PINK1 (all R&D systems). In a separate reaction 0.75 µM Parkin(Ubiquigent) was preincubated with 20 µM compound 4 (or equal volume ofDMSO). Both reactions were diluted in Ub buffer (final concentration: 20mM HEPES pH 7.2, 10 mM MgCl₂, 0.1 mM EGTA, 500 µM TCEP and 10% ATPregeneration system (20 mM HEPES pH 7.6, 10 mM ATP, 300 mMphosphocreatine, 10 mM MgCh, 10% glycerol, 1.5 mg/mL creatinephosphokinase (all Sigma Aldrich))). 1U Apyrase (Sigma) was added per 9µL reaction and both reactions were combined and incubated for anadditional 30 min. 100 µL of 1xLDS buffer was added per 10 µL reactionand samples were split for -/+ DTT (20 mM final).

To provide substrates for Parkin ligase activity, mitochondrialfractions for an ex cellulo ubiquitylation assay were provided. Thesesamples were prepared from HeLa cells that express PINK1, but lackParkin and that had been left untreated or were treated with 10 µM CCCPfor 2 h. Aliquots were resuspended in reactions containing recombinantpurified E1, E2, Ub and Parkin as well as ATP and either compound 4 orDMSO as a control. Mono-ubiquitylation of MFN1 was observed in theabsence of compound, but poly-ubiquitylation was robustly induced whencompound 4 was added to the reaction, especially at shorter time points(FIG. 15E). Notably, there was no effect when mitochondria were isolatedfrom cells that were left untreated (i.e., no PINK1).

The in vitro assays with mitochondrial preparations were performed asfollows. HeLa cells were either left untreated or treated with 10 µMCCCP for 2 h. Cells were harvested in solution B (20 mM HEPES pH 7.6,220 mM mannitol, 70 mM sucrose, 10 mM KAc containing EDTA-free Completeand PhosStop) and homogenized by 10 strokes through a 23G needlefollowed by 10 strokes through a 27G needle. Lysates were centrifugedfor 5 min at 800 g and supernatant spun for 20 min at 8000 g to pelletmitochondria. The mitochondrial pellet was resuspended in solution B andthe protein concentration determined by BCA. Aliquots of 50 µg wereprepared, spun at 20,000 g for 5 min, the supernatant removed and storedat -80° C. The in vitro reaction was prepared in Solution B buffer andcontained 45 µM Ub, 0.1 µM GST-E1, 1 µM E2/UbcH7, 2 mM DTT and 1 µL ofATP regeneration system per 10 µL reaction. Reactions with 0.75 µMParkin were prepared as one mix that was split in different reactionsbefore compound (20 µM) or DMSO was added. Reactions were incubated at37° C. for 2 h and 10 µL were added per 50 µg mitochondria. Samples wereincubated at 30° C. for the indicated time points, centrifuged, washedonce with solution B before mitochondrial pellet was resuspended in 100µL LDS buffer containing 100 mM DTT and 1% Triton X-100. The supernatantof the reaction (8 µL) was saved and mixed with 32 µL 2x LDS buffer.Samples were heated to 35° C. shaking for 30 min before gel loading.

To validate direct binding of activating compounds to Parkin aspredicted from the in silico approach, a thermofluor shift assay wasused. Recombinant purified Parkin was mixed together with compound 4 orDMSO as a control and the melting temperature was monitored using SYPROOrange dye. Addition of compound 4 significantly elevated the meltingtemperature of Parkin (FIGS. 15G-15H) suggesting the direct associationof Parkin and drug. This result, together with the cell culture data andin vitro experiments corroborate that PINK1-dependent priming of Parkinis required for compound activity as predicted from the MDS.

The thermal shift assay was performed as follows. Per sample (5 µL), 50ng of Parkin (Ubiquigent) was mixed with 0.5 µL 1/50 diluted SYPROOrange (Invitrogen), 0.5 µL 10x buffer (200 mM HEPES pH 7.6, 100 mMMgCl₂, 20 mM DTT and 1 mM EGTA) and 0.5 µM compound or equivalentamounts of DMSO. Samples were run in opaque 384-well plates on aLightCycler 480 system (Roche Applied Science) in a melt curve analysiswith 10 acquisitions per °C. Data was exported and analyzed using theProtein Melting Analysis tool (Roche Applied Science).

Example 12: Parkin Binding to Ubiquitin

HeLa 3xFLAG-Parkin C431S cells were seeded in 6-well plates and allowedto attach overnight. Cells were treated with 1 µM of compounds 5, 21-28,32, 33, or 43-46 2 h before adding CCCP for another 2 h. 10 µM CCCP wasadded to positive control (PC) wells, 3.5 µM CCCP to compound (+) andnegative control (NC) wells. Some samples did not receive CCCP (-).Cells were harvested in boiling hot SDS lysis buffer and proteinconcentration was determined by BCA. Samples were split and left eitheruntreated or were treated with NaOH as indicated. Samples were run on an8-16% Tris-Glycine gel, blotted onto membranes and probed withantibodies against Flag. Vinculin served as a loading control. Bandshift indicates Parkin binding to Ubiquitin, which is cleavable withNaOH (FIG. 21 ).

Incorporation by Reference

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

Equivalents

The disclosure can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the disclosure described herein. Scope of thedisclosure is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-39. (canceled)
 40. A compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: A is CH, N, orS; B is CH, N, O, or S; D is C or N; E is CH or N; L is C₁₋₃ alkylene orC(═O); W is CH, CH₂, N, or NR^(a); X is CH, CH₂, N, or NR^(b); Y is CH,CH₂ or O; Z is N or CR^(2'); R¹ is H or C₁-₃ alkyl; R² is absent or C₁₋₆alkylene; R^(2') is H or C₁₋₆ alkyl; or, when Z is CR^(2'), R² andR^(2'), together with C, can be taken together to form a C₁₋₆heterocyclic ring; R³ is H, halogen, C₁₋₃ alkyl, C₃₋₆ cycloalkyl, orC₁₋₃ alkoxy; R⁴ is H or C₁₋₃ alkyl; R^(4') is H or C₁₋₃ alkyl; R^(a) isH or C₁₋₃ alkyl; R^(b) is C₁₋₃ alkyl; m is 0, 1 or 2; n is 1 or 2; p is0 or 1; and the dashed lines can be single or double bonds; with theproviso that when n is 2, R¹ is methyl, D is C, R³ is H, and A, B, and Eare all CH, then at least one of W, X, and Y is not CH₂. 41-45.(canceled)
 46. The compound of claim 40, wherein D is C. 47-49.(canceled)
 50. The compound of claim 40, wherein L is methylene.
 51. Thecompound of claim 40, wherein W is CH₂.
 52. The compound of claim 40,wherein W is N. 53-58. (canceled)
 59. The compound of claim 40, whereinZ is N.
 60. The compound of claim 40, wherein R² is absent. 61-62.(canceled)
 63. The compound of claim 40, wherein R¹ is H.
 64. Thecompound of claim 40, wherein R¹ is methyl. 65-66. (canceled)
 67. Thecompound of claim 40, wherein R³ is H, halogen, or C₃-₆ cycloalkyl.68-69. (canceled)
 70. The compound of claim 40, wherein R⁴ and R^(4')are each H.
 71. (canceled)
 72. The compound of claim 40, wherein n is 1.73. The compound of claim 40, wherein n is
 2. 74. The compound of claim40, wherein m is 1 or
 2. 75. The compound of claim 40, wherein p is 0.76. The compound of claim 40, wherein p is
 1. 77. The compound of claim40, wherein the compound of Formula II is selected from the groupconsisting of:

and

or a pharmaceutically acceptable salt thereof.
 78. The compound of claim40, wherein the compound of Formula II is a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof, wherein: A is CH or N; Bis N, O, or S; D is C or N; E is CH or N; W is CH₂ or NR^(a); X is CH,CH₂, N, or NR^(b); Y is CH, CH₂ or O; L is C₁₋₃ alkylene or C(═O); R¹ isH or C₁₋₃ alkyl; R³ is C₁₋₃ alkyl or C₁₋₃ alkoxy; R⁴ is H or C₁₋₃ alkyl;R^(4’) is H or C₁₋₃alkyl; R^(a) is H or C₁₋₃ alkyl; R^(b) is C₁₋₃ alkyl;n is 1 or 2; m is 0, 1 or 2; p is 0 or 1; and the dashed lines can besingle or double bonds.
 79. The compound of claim 40, wherein thecompound of Formula II is a compound of Formula IIb:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is C_(1–3)alkyl; and R⁵ is (C₁₋₃ alkyl)phenyl.
 80. A pharmaceutical compositioncomprising a compound of claim 40, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable carrier.
 81. Amethod of activating the enzymatic activity of an E3 ubiquitin ligase ina subject, the method comprising administering to the subject atherapeutically effective amount of a compound of claim 40, or apharmaceutically acceptable salt thereof. 82-91. (canceled)
 92. A methodof treating a disease or disorder associated with diminished E3ubiquitin ligase enzymatic activity in a subject, the method comprisingadministering to the subject a therapeutically effective amount of acompound of claim 40, or a pharmaceutically acceptable salt thereof. 93.The method of claim 92, wherein the E3 ubiquitin ligase is selected fromthe group consisting of Parkin, ARIH1 (HHARI), ARIH2 (TRIAD1), RNF31(HOIP), RBCK1 (HOIL-1L), MUL1 (MAPL, MULAN), MARCH5 (MITOL), E3A, mdm2,anaphase-promoting complex (APC), UBR5 (EDD1), SOCS, LNXp80, CBX4,CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3,HERC4, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3,PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A,UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, and WWP2.
 94. (canceled)95. The method of claim 92, wherein the disease or disorder isParkinson’s disease. 96-114. (canceled)